# Conserved Microsynteny ofNPR1 with Genes Encoding  a Signal Calmodulin-Binding Protein and a CK1-Class Protein Kinase in Beta vulgaris and Two Other Eudicots

Authors: David Kuykendall; Jonathan Shao; Tammy  Murphy
Journal: International Journal of Plant Genomics
 (2008)

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## Abstract

NPR1 is a gene of central importance in enabling plants to resist microbial attack. Therefore, knowledge of nearby genes is important for genome analysis and possibly for improving disease resistance. In this study, systematic DNA sequence analysis, gene annotation, and protein BLASTs were performed to determine genes near the NPR1 gene in Beta vulgaris L., Medicago truncatula Gaertn, and Populus trichocarpa Torr. & Gray, and to access predicted function. Microsynteny was discovered for NPR1 with genes CaMP, encoding a chloroplast-targeted signal calmodulin-binding protein, and CK1PK, a CK1-class protein kinase. Conserved microsynteny of NPR1, CaMP, and CK1PK in three diverse species of eudicots suggests maintenance during evolution by positive selection for close proximity. Perhaps close physical linkage contributes to coordinated expression of these particular genes that may control critically important processes including nuclear events and signal transduction.


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## Body

## 1. Introduction

Research done onArabidopsis thaliana (L.) Heynth over a 10-year
period in a number of laboratories has amassed considerable evidence that the NPR1 gene (also called NIM1) is of central importance in
determining the plant ability to resist microbial attack [1]. In essence, global
plant defense responses to pathogen invasion are controlled by the NPR1 gene product and intracellular
redox state, since an inactive dimeric NPR1 protein in the cytosol is reduced to
the active monomer which then migrates to the nucleus and activates expression
of pathogen-induced “pathogenesis-related” (PR) genes [2].The central role of NPR1 in positively activating
defense mechanisms in response to biotic stress suggests the possibility of
enhancing disease resistance in plants by genetic manipulation of theNPR1 gene. In fact, an increasing number
of attempts to improve disease resistance in plants by modifying expression of NPR1 have been reported [3–7].Microsynteny is genomics information that can be used
to predict the location of homologous genes in different species.  Knowledge of microsynteny of genes colinear withNPR1 in crop species could perhaps be
used to devise innovative strategies for molecular genetic modification in
order to improve disease resistance. As a step towards identifying genes located
near the NPR1 gene in sugarbeet, a bacterial
artificial chromosome (BAC) library [8] was screened and an NPR1-carrying clone, SBA091H24, was identified
[9]. The B. vulgaris BvNPR1 gene encodes a predicted protein
product being 100% identical to that deduced from the sequence of the cDNA for B. vulgaris NPR1
(GenBank accession AY640381). SMART analysis of the predicted BvNPR1 gene product [9] showed a BTB/POZ
domain and two ARDs, or ankyrin repeat domains [10], both being characteristic
of NPR1 proteins and other transcriptional activators within the nucleus. NPR1 is responsible for disease
resistance priming or “induced resistance,” a result of coordinated expression
of multiple defense mechanisms/pathways to effectively resist microbial attack [11].TheNPR1 gene in sugarbeet has been only recently shown [12] to be essential for induced
systemic resistance, as in the model A.
thaliana (L.) Heynth [1]. In both
plant species, the activated form of NPR1 migrates into the nucleus and activates
the transcription of genes involved with resisting disease-forming microbes [12].Conservation of microsynteny among distinct
families of eudicots was discovered inLycopersicon
esculentum Mill. (tomato) and A. thaliana, where large-scale
duplications followed by selective gene loss have created a network of chromosomal
synteny [13]—an accepted
paradigm. By developing physical genetic maps based on expressed sequence tags (ESTs),
Dominguez et al. [14] discovered conserved synteny with Arabidopsis among the genomes of four phylogenetically divergent
eudicot crops, namely, sugarbeet, potato, sunflower, and plum.In this study, complete
BAC sequence analysis identified two core plant genes tightly physically linked
toNPR1, and established a conservation
of microsynteny between the NPR1 gene
regions of sugarbeet and two other eudicot species. We report the gene content
and organization of a 130 Kb DNA contig (continuous fragment) from an NPR1-carrying sugarbeet BAC. Comparison of similar NPR1-carrying DNA contigs from M.
truncatula and P. trichocarpa showed that orthologs of genes encoding NPR1,
a signal-peptide calmodulin-binding protein (CaMP) and a CK1-class dual-specificity
protein kinase (CK1PK) occur in the same order and with a conserved direction
of transcription in three divergent species of eudicots. This suggests positive natural selection for maintaining the physical proximity of
genes whose products control certain essential nuclear events and a particular signal
transduction function, as yet undefined.


## 2. Materials and Methods

### 2.1. DNA Sequencing

Genomic DNA ofB. vulgaris hybrid US H20 [15], with an estimated 750 Mb genome size, had previously
been used to construct a BAC library by ligating large DNA fragments resulting
from partial HindIII digestion into
vector pECBAC1 [16]. About 34,500 clones comprised the BAC library, and the average
insert size was about 120 Kb, providing about 6.1X genome coverage [8]. Primers
designed on the basis of data extracted from GenBank accession  AY640381,
a cDNA sequence for B. vulgaris NPR1, were utilized to screen and
identify a BvNPR1-carrying BAC [9]. The
presence of a complete genomic BvNPR1 gene was established by partial DNA sequence analysis of BAC clone SBA091H24 (GenBank
accession DQ851167) [9].BAC sequencing was completed at Washington University’s
Genome Sequencing Center in St. Louis, Mo, USA
(http://genome.wustl.edu/). The BAC clone SBA091H24
was provided to the Genome Sequencing Center
as a glycerol stock. Purification, library construction, shotgun cloning, and
sequence analysis were performed on a sufficient number of random subclones to
provide about 9.4X coverage. ABI 3730 capillary sequencers were used. Data
was assembled using the phred/phrap suite (http://www.phrap.org/).


### 2.2. Gene Annotation

Analysis of the sequence data was performed using Lasergene (DNASTAR, Inc., Madison, WI, USA)
for assembly and NCBI BLAST [17]. The sequence contig was screened for coding
sequence using a combination of the following programs: GeneMark [18, 19] for
eukaryotes (http://exon.gatech.edu/GeneMark/eukhmm.cgi), GenScan (http://genes.mit.edu/GENSCAN.html),
FGENESH (http://softberry.com/), and GRAIL (http://grail.lsd.ornl.gov/grailexp/). In
all cases, A. thaliana was chosen as
a model, and default settings were used. BLASTP searches were performed at
the National Center for Biotechnology Information (NCBI)
website (http://www.ncbi.nlm.nih.gov/BLAST/).  Percent
identities and percent similarities were obtained using BLAST alignments. Manual curation of proteins was performed
using Lasergene MegAlign and EditSeq sequence analysis software, where
applicable simple modular architecture research tool (SMART) [20]
database (http://smart.emblheidelberg.de/) and Motif
Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan)
were used to identify protein domains and motifs, respectively. ARTEMIS (http://www.sanger.ac.uk/Software/Artemis/) was used to
collate data and facilitate annotation.Calmodulin target database (http://calcium.uhnres.utor-onto.ca/ctdb/ctdb/home.html)
was used to identify the most likely calmodulin-binding site in a peptide
sequence. The hmmtop server (http://www.enzim.hu/hmmtop/) was used to predict transmembrane helices,
and a signal peptide was detected using the SignalP 3.0 and TargetP 1.0 servers (http://www.cbs.dtu.dk/services/).BAC chromosome and genomic annotation datasets forM. truncatula and O. sativa L. were accessed through TIGR (http://www.tigr.org/). P. trichocarpa (black cottonwood) genome
information [21] was accessed through NCBI (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gen-omeprj&cmd=Retrieve&dopt=Overview&list_uids=10770).


### 2.3. Identification of Colinearity

BLAST searches were performed for protein products of the predicted ORFs
of theB. vulgaris NPR1 BAC against databases
for A. thaliana, M. truncatula, P. trichocarpa, and O. sativa
L. ssp. japonica. High-scoring pairs (HSPs), the predicted
protein products with highly significant matches, were considered as products
of orthologous genes. Corresponding DNA regions are considered to be microcolinear
when two or more orthologous genes are present in physical proximity, in the
same order, and are transcribed in the same direction.GenBank accession NC_008472.1 is representative of
linkage group VI ofP. trichocarpa [21], and the subsets
of particular interest were coordinates from 8404895 to 8453314. GenBank accession AC124609 represents BAC clone mth229b13, a subset of
chromosome 2 of M. truncatula (http://www.tigr.org/tigr-scripts/medicago/contig_location_association.pl?chromosome=2)
[22, 23]. AC124609 (1...61740 bases) was extracted and compared to our B. vulgaris GenBank accession EF101866
(1...129695 bases).


## 2.1. DNA Sequencing

Genomic DNA ofB. vulgaris hybrid US H20 [15], with an estimated 750 Mb genome size, had previously
been used to construct a BAC library by ligating large DNA fragments resulting
from partial HindIII digestion into
vector pECBAC1 [16]. About 34,500 clones comprised the BAC library, and the average
insert size was about 120 Kb, providing about 6.1X genome coverage [8]. Primers
designed on the basis of data extracted from GenBank accession  AY640381,
a cDNA sequence for B. vulgaris NPR1, were utilized to screen and
identify a BvNPR1-carrying BAC [9]. The
presence of a complete genomic BvNPR1 gene was established by partial DNA sequence analysis of BAC clone SBA091H24 (GenBank
accession DQ851167) [9].BAC sequencing was completed at Washington University’s
Genome Sequencing Center in St. Louis, Mo, USA
(http://genome.wustl.edu/). The BAC clone SBA091H24
was provided to the Genome Sequencing Center
as a glycerol stock. Purification, library construction, shotgun cloning, and
sequence analysis were performed on a sufficient number of random subclones to
provide about 9.4X coverage. ABI 3730 capillary sequencers were used. Data
was assembled using the phred/phrap suite (http://www.phrap.org/).


## 2.2. Gene Annotation

Analysis of the sequence data was performed using Lasergene (DNASTAR, Inc., Madison, WI, USA)
for assembly and NCBI BLAST [17]. The sequence contig was screened for coding
sequence using a combination of the following programs: GeneMark [18, 19] for
eukaryotes (http://exon.gatech.edu/GeneMark/eukhmm.cgi), GenScan (http://genes.mit.edu/GENSCAN.html),
FGENESH (http://softberry.com/), and GRAIL (http://grail.lsd.ornl.gov/grailexp/). In
all cases, A. thaliana was chosen as
a model, and default settings were used. BLASTP searches were performed at
the National Center for Biotechnology Information (NCBI)
website (http://www.ncbi.nlm.nih.gov/BLAST/).  Percent
identities and percent similarities were obtained using BLAST alignments. Manual curation of proteins was performed
using Lasergene MegAlign and EditSeq sequence analysis software, where
applicable simple modular architecture research tool (SMART) [20]
database (http://smart.emblheidelberg.de/) and Motif
Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan)
were used to identify protein domains and motifs, respectively. ARTEMIS (http://www.sanger.ac.uk/Software/Artemis/) was used to
collate data and facilitate annotation.Calmodulin target database (http://calcium.uhnres.utor-onto.ca/ctdb/ctdb/home.html)
was used to identify the most likely calmodulin-binding site in a peptide
sequence. The hmmtop server (http://www.enzim.hu/hmmtop/) was used to predict transmembrane helices,
and a signal peptide was detected using the SignalP 3.0 and TargetP 1.0 servers (http://www.cbs.dtu.dk/services/).BAC chromosome and genomic annotation datasets forM. truncatula and O. sativa L. were accessed through TIGR (http://www.tigr.org/). P. trichocarpa (black cottonwood) genome
information [21] was accessed through NCBI (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gen-omeprj&cmd=Retrieve&dopt=Overview&list_uids=10770).


## 2.3. Identification of Colinearity

BLAST searches were performed for protein products of the predicted ORFs
of theB. vulgaris NPR1 BAC against databases
for A. thaliana, M. truncatula, P. trichocarpa, and O. sativa
L. ssp. japonica. High-scoring pairs (HSPs), the predicted
protein products with highly significant matches, were considered as products
of orthologous genes. Corresponding DNA regions are considered to be microcolinear
when two or more orthologous genes are present in physical proximity, in the
same order, and are transcribed in the same direction.GenBank accession NC_008472.1 is representative of
linkage group VI ofP. trichocarpa [21], and the subsets
of particular interest were coordinates from 8404895 to 8453314. GenBank accession AC124609 represents BAC clone mth229b13, a subset of
chromosome 2 of M. truncatula (http://www.tigr.org/tigr-scripts/medicago/contig_location_association.pl?chromosome=2)
[22, 23]. AC124609 (1...61740 bases) was extracted and compared to our B. vulgaris GenBank accession EF101866
(1...129695 bases).


## 3. Results

A 130 Kb BAC designated SBA091H24, containingB. vulgaris chromosomal DNA, more specifically the sugarbeet NPR1 gene [9], was sequenced and fully annotated
(GenBank accession EF101866). The bioinformatics
tools FGENESH, GeneMark, GenScan, and GRAIL were used as gene finders. Predicted
gene names and predicted functions of deduced amino acid sequences, where
possible, are presented in Table 1, and a visual representation of exon
structure is shown in Figure 1. On the 130 Kb contig, a total of 17 potential open
reading frames (ORFs), or protein coding regions, were identified. Only four out
of the 17 open reading frames (ORFs) were predicted to produce protein products
with high amino acid similarity to known products of core plant genes (Table 1). In addition to four core plant genes, the 130 Kb contig contains five retrotransposon genes, a transposon gene, and seven other
genes whose products lacked a predicted function.Table 1
Predicted genes and their designated functions.
GeneProduct length (a.a.)Best BLAST HitaE-valueSimilarityDesignationbHp1259ABE851185.0E−0849/101HypotheticalcHSF (similar to HSFA9)337AAM438043.4E−47185/320HSF transcription factorNPR1604AAT576400.0E+00604/604NPR1 disease resistanceHp2259noneHypotheticalcIntegrase1516ABE910910.0E+00903/1535Integrase (copia type)Hp3403ABD832801.1E−124315/325UnknowndReverse transcriptase (RT)1501ABE833030.0E+00996/1503Gypsy-type retrotransposonHp4302CAH671201.0E−1378/177HypotheticalcHp5263ABD833011.0E−0941/63HypotheticalcHp6222noneHypotheticalcHp7593ABD832804.1E−60186/282UnknowndORF1 ofCoe1 (a composite of class I and class 2 elements) [25]188NP_199616.11e−17106/192Hypothetical (retroelement-like
gene)Coe1 (transposon
gene)1399ABE828480.0E−00604/956TransposonORF2 ofCoe1 (an integrase pseudogene)297ABA956774.0E−70177/298Rvt2 domain integrase pseudogeneCalmodulin-binding protein (CaMP)525NP_9746737.0E−161361/495Signal-peptide calmodulin-binding
proteinRT (reverse transcriptase)198ABF814173.0E−37126/213RT-like geneCK1-class protein kinase
(CK1PK)473BAB923460.0E+00411/476CK1-class dual-specificity
protein kinaseaGenBank accession number or protein ID of the best
BLAST hit, followed by the E-value and percent (similar/total amino acids)
similarity between the query and the best hit.
bDesignation based on the deductions possible via
bioinformatics tools listed in Section 2. Functional classification based on
the result of protein BLAST search.
cNA: not applicable; putative function of the product was not
identified.
dSimilar to FGENESH 21 [26].Figure 1
Schematic representation of genes
annotated on the 130 Kb genomicNPR1-carrying BAC from sugarbeet (GenBank accession EF101866)
based on FGENESH, GenScan, GeneMark, and GRAIL gene finders as well as SMART
and Motif Peptide Scan results. (Blue:
core plant genes involved with nuclear events/signal transduction/defense; yellow: transposon; orange:
retrotransposons; red: encode only hypothetical or
unknown proteins). The predicted genes begin with a bar and end with an
arrowhead, thus indicating the direction of transcription.In addition
toNPR1, another core plant gene,
predicted on the 130 Kb sugarbeet DNA contig from BAC clone SBA091H24, was
composed of two exons that encode a heat shock factor (HSF) protein with a
conserved DNA binding domain  (E=2e−29) from amino acid positions 45 to 205. This HSF gene is located between the NPR1 gene and gene Hp1, encoding a hypothetical protein with some similarity to
retrotransposon-encoded proteins. The HSF gene and NPR1 are transcribed in opposing directions. The predicted HSF protein
has moderately high similarity (Table 1) to the protein product of HSFA9—a leaf pattern
morphogenesis-controlling gene of sunflower, Helianthus annuus [24].Beginning at about 70 Kb upstream ofNPR1, another
core plant gene encodes a calmodulin-binding
protein (CaMP) that, as SMART analysis revealed, has an IQ domain from amino
acid positions 131 to 153. Motif Scan suggested
(1) an IMP dehydrogenase/GMP reductase domain at amino acids 121–444, (2) several protein
kinase phosphorylation sites, and (3) an involucrin repeat at amino acids 206–215. The predicted protein product gave numerous
BLAST hits to calmodulin-binding proteins. Calmodulin target database predicted
amino acid positions 201–209 as the most
likely calmodulin-binding site. Transmembrane helices were predicted at amino acid positions 337–353 by the hmmtop
server, and the SignalP 3.0 server predicted a cleavage site in the N-terminal signal-peptide
sequence between glutamine and lysine in amino acid positions 17 and 18.  Thus, we deduced that the mature CaMP protein is
508 amino acids in length.  The pro-CaMP
was predicted as 525 amino acids in length, and the presence of a membrane-targeted
N-terminal signal-peptide sequence suggests that peptide maturation occurs upon
passage into or through a membrane. TargetP predicted that the targeted sites
of subcellular localization of mature protein were chloroplasts.Beginning at about 90 Kb upstream
ofNPR1, a third core plant gene  encodes a “casein kinase class 1” protein
kinase (CK1PK), identified by numerous BLAST hits with E-values of 0 to CK1PKs.
SMART analysis revealed a dual-specificity STYKc protein kinase domain from amino acids 9–211. This type of kinase phosphorylates either
serine, threonine, or tyrosine residues.Conservation of microsynteny was discovered inB. vulgaris, M. truncatula, and P. trichocarpa (Figure 2), but not in A. thaliana or O. sativa L., by comparative DNA analysis of three core plant
genes: (1) a CK1 class dual-specificity protein kinase gene, (2) a signal
calmodulin-binding protein gene, and (3) the disease resistance-priming NPR1 gene. The high degree of amino acid
similarity as well as identity of the predicted products of these respective microcolinear
genes (Table 2) clearly indicates that they are orthologous gene pairs with shared
functions.Table 2
Percent
amino acid identity and similarity exhibited by amino acid alignments  of
the conserved products of orthologous gene pairs.
OrthologousB. vulgaris NPR1B. vulgaris CaMPB. vulgaris CK1PKgene pairIdentity/similarityIdentity/similarityIdentity/similarityMedicago homologs60%/74%57%/68%67%/77%Populus
homologs65%/79%56%/68%75%/82%Medicago NPR1Medicago CaMPMedicago CK1PKIdentity/similarityIdentity/similarityIdentity/similarityPopulus
homologs62%/75%60%/71%69%/78%Figure 2
Schematic representation of the microcolinearity found betweenNPR1-carrying regions of (a) B. vulgaris (GenBank EF101866),
(b) M. truncatula (GenBank AC124609.20), and (c) a subset of P. trichocarpa
(NC_008472.1). Genes are indicated by boxes: NPR1 (gray box); CaMP encoding
a calmodulin-binding protein (black box); specifying a CK1PK-class protein kinase (white box). Arrows indicate the
direction of transcription. The gray bars indicating the distances in Kb
between genes are from the end exon of one gene to the end exon of the next
gene. The length of the DNA regions under comparison is indicated in base
pairs. As of this writing, the TIGR annotated NPR1 region for M. truncatula is a draft; shown is only about one half of a BAC sequence since there was a gap
in the sequence near the center.The three orthologous
gene pairsNPR1, CaMP, and CK1PK are
colinear, that is, with both the same gene order and direction of transcription, in B. vulgaris, M. truncatula,
and P. trichocarpa (Figure 2). In particular, in the comparison of presumptive orthologous
gene pairs in B. vulgaris and M. truncatula, the NPR1, CaMP, and CK1PK genes
encode proteins that have amino acid identities of about 60%, 57%, and 67%, and that
exhibit amino acid similarities of about 74%, 68%, and 77%, respectively (Table 2). Similarly, in the comparison of presumptive orthologous gene pairs
in B. vulgaris and P. trichocarpa, the three
orthologous gene pairs, (NPR1, CaMP, and CK1PK), produce protein
products with amino acid identities of about 65%, 56%, and 75% and amino acid
similarities of about 79%, 68%, and 82%, respectively (Table 2). Finally, comparison
of the amino acid identities and similarities of the predicted products of the NPR1, CaMP, and CK1PK
genes in P. trichocarpa and M. truncatula (Table 2) indicates conserved orthologous gene
pairs.As mentioned above, about
2 Kb downstream ofNPR1 in B. vulgaris, a fourth
core plant gene (Figure 2) was predicted to produce a protein product with
moderately high similarity to that produced by the embryonically expressed heat
shock factor (HSF) A9 gene in
sunflower. Microcolinearity of this particular HSF gene with NPR1 did
not occur in M. truncatula. On the
other hand, NPR1 in P. trichocarpa is separated from an HSF gene by only 1 Kb, but it encodes a protein that has only about
39% amino acid identity and 54% amino acid similarity with the protein encoded
by the HSF gene adjacent to the NPR1 gene in B.
vulgaris. Thus, the structure and function
of the respective HSF proteins is not as highly conserved as were the three
other colinear core plant genes in poplar and sugarbeet.Comparison of theArabidopsis NPR1 region with
those of M. truncatula, P. trichocarpa, and B. vulgaris revealed that Arabidopsis lacks conserved microsynteny
of CaMP and CK1PK genes with NPR1. In O. sativa
L., the NPR1 genomic region has a
gene encoding a calmodulin-binding protein, but it is transcribed in the same
direction as NPR1 rather than in opposing
directions as in B. vulgaris and M. truncatula; also the
CK1PK gene, most proximal to NPR1 in O. sativa L., is greater than 250 Kb
away (not shown). Thus, microsynteny, as in three
out of four eudicot species, did not occur in O. sativa L., perhaps not unexpectedly as it is a monocot.The six other noncore genes were retrotransposon or DNA transposon ones. Four putative genes were predicted to encode proteins with strong BLAST hits to retrotransposons or retrotransposon-like genes. An integrase gene has four exons and, from amino acid positions 750 to 900, this ORF encodes anrve core domain (E=3e−30), being a characteristic of integrases. With the integrase gene and the downstream putative gene Hp3 being combined, both share about 98% nucleotide identity (BLASTN) with highly related genes on a 9 Kb DNA contig (GenBank accession ABD83280) of BAC62 from sugarbeet chromosome  9 [26]. A putative reverse transcriptase gene about 5 Kb downstream of Hp3 consists of a single exon encoding a polyprotein with Rvt2 RNA-dependent DNA polymerase domain (E=7e−24), an rve integrase domain (E=6e−21), and a poorly conserved (E=8e−10) gag capsid-like protein domain.Coe1, previously identified by our group using LTR_STRUC analysis as a novel
composite of class I and class II elements [25], has three genes.
Briefly, one gene has a single exon ORF1 (Table 1), encoding a retroelement-like protein. ORF1 is about 2 Kb upstream of a gene that encodes a DNA
transposase since its predicted product produced many significant BLAST alignments with DNA
transposases, with E-values equal to 0. The very highly conserved transposase family tnp2
domain (E=1e−94) occurs in amino acid
residue positions 200–400 of the predicted protein. Coe1’s transposase gene is also flanked
by ORF2, a pseudogene of a copia-like integrase, and all three genes are within LTRs [7].
ORF2 is 2 Kb downstream of the tnp2-class DNA transposase gene, and its predicted product showed, by BLAST analysis,
a significant alignment with several putative retrotransposon proteins from wine grape and rice; for example, it aligned to O. sativa’s putative
Ty1-copia subclass retrotransposon (accession ABA95677) with E-value of ≤e−50, 38% amino acid identity,
and 49% amino acid similarity.Downstream of the gene  encoding a calmodulin-binding protein, a putative reverse transcriptase
gene encoded a predicted protein with only a moderate BLAST alignment (E=3e−37)
to a reverse transcriptase Rvt-2 domain found in a novel retrotransposon-like TIR-NBS-LRR-type
disease resistance protein in P. trichocarpa Torr. & Gray (Table 1),
and the similarity corresponds to a shared Rvt-2-type domain.InM. truncatula, a TIR-NBS-LRR-type resistance gene occurs downstream of NPR1 (not shown). The predicted product
of the M. truncatula resistance gene analogue
(RGA) near NPR1 has 39% amino acid
identity and 59% amino acid similarity with the Gro1-4 nematode resistance gene of Solanum
tuberosum (GenBank accession AY196152.1).Comparative
sequence analysis and gene annotation inB.
vulgaris, M. truncatula, and P. trichocarpa revealed that these three
divergent species of eudicots exhibit conserved microsynteny of genes that encode
the centrally important disease resistance priming NPR1, a signal-peptide calmodulin-binding protein (CaMP), and a
CK1-class dual-specificity protein kinase (CK1PK). The orthologs occur in the
same order and with the same direction of transcription.


## 4. Discussion

In this study, comparison of
orthologousNPR1 gene regions of B. vulgaris, M. truncatula, and P. trichocarpa revealed for
the first time conserved microsynteny of the defense-priming NPR1 gene with a CaMP gene, encoding a calmodulin-binding protein, and with a CK1PK gene, specifying a CK1-class dual-specificity
protein kinase.Calmodulin-binding proteins in
plants are very diverse, exhibit various motifs, and perform a correspondingly
wide variety of functions [27–30]. For example, an Arabidopsis ethylene-upregulated calmodulin-binding protein triggers senescence and death [31].Calcium and calmodulin mediate
a complex signal transduction network in plants through protein kinases (PKs),
and some PKs are unique to plants [32]. They are literal “hubs” of sensory perception and signal
transduction. Examples include a calmodulin-binding
PK in Arabidopsis that negatively
regulates tolerance to osmotic stress [32] and a calmodulin-binding PK in
tobacco (Nicotiana tabacum L.), involved with negative regulation of
flowering [33].It seems
reasonable to hypothesize that theCaMP gene product, which is a chloroplast-targeted,  signal-peptide, calmodulin-binding
transmembrane protein, could play a role in rapid
activation of a defense cascade during either general stress or pathogen
response. Just as the NPR1 gene is critical for disease resistance priming in plants, some
calmodulin-binding proteins are pathogenesis-related. For example, de novo synthesis of a
calmodulin-binding peptide with a DNA-binding domain at the N-terminus is
induced by ethylene formed by the plant in response to wounding and/or
infection [34]; also in Arabidopsis, another
calmodulin-binding protein is  pathogenesis-related as well [35]. Moreover, in Glycine max L. (soybean), specific
calmodulin isoforms are required for the expression of pathogen-induced proteins
upregulated by the NPR1 disease
resistance control gene [36]. Conserved
microsynteny of CK1PK, CaMP, and NPR1 genes, discovered in three out of
four eudicot species examined, could be hypothesized to  suggest that their
protein products play essential cellular roles related to plant
defense response.We propose a new hypothesis that conserved gene microsynteny of certain core
plant genes in eudicots may correlate with either similar subcellular
localization or with similar function. Either possibility for the protein
products of the three core plant genes herein described is plausible. Activated monomeric NPR1 functions in the plant
nucleus, where CK1PKs are also localized. CK1s are believed to control
circadian rhythm [37] and chromosome partitioning during meiotic cell division [38]
in all eukarotic cells. It should be noted that in O. sativa a novel
family of dual-specificity PKs is involved in controlling the plant responses
to biotic as well as abiotic stresses [39].  Based on available literature [30, 31, 39], the CK1PK gene localized near NPR1 in certain eudicots may play a role
in controlling the expression of stress-responsive genes in plants.A total of
11 retrotransposons (RTs), DNA transposons, and hypothetical genes lie within
the approximately 80 Kb stretch of DNA between theNPR1 and CaMP genes in B. vulgaris; therefore, we conclude that this
region is rich in repetitive elements, and several insertions of mobile genetic
elements have occurred during evolutionary divergence (Kuykendall et al.,
unpublished). ORFs,
originating from either retrotransposons or viruses, from DNA transposons and
other repetitive elements need not be considered disruptive of the conservation
of colinearity when the core genes nevertheless occur in physical proximity, in
the same order, and are transcribed in the same relative direction. Fortunately,
our approach was not to be dissuaded by the repetitive elements that occur between NPR1 and CaMP in B. vulgaris comparing
the orthologous regions of M. truncatula and P. trichocarpa, and thus we
discovered conserved microsynteny for orthologous NPR1, CaMP, and CK1PK genes in these three
eudicot species. This conserved microsynteny of NPR1, CaMP, and CK1PK in B. vulgaris, M. truncatula, and P. trichocarpa likely has an evolutionary basis through a yet
undefined selective advantage.The observed conservation of microsynteny of the three core plant
genes could be hypothesized to result, in part, from positive natural selection
for physical proximity. Hypothetically, close physical linkage may facilitate
coordinated expression of genes critical in certain controlling nuclear
events, such as those for which the protein products ofCK1PK and NPR1 are known,
or are responsible, either directly or indirectly, for the initiation of a dynamic
signal transduction cascade as can be hypothesized for the predicted product of CaMP.Reverse transcriptase (RT) PCR expression studies will be useful in
determining whether theCaMP or CK1PK is upregulated in response to the
administration of factors which induce the production of either pathogenesis-related
or stress-related proteins. Such studies will hopefully allow one to determine whether the
chloroplast-targeted signal-peptide calmodulin-binding protein gene (CaMP) or the nuclearly localized casein
kinase 1 protein kinase gene (CK1PK) may play a role(s) in pathogen and/or stress response.In summary, in addition toNPR1 gene and the CaMP and CK1PK genes herein described for
the first time, the 130 Kb NPR1-carrying B. vulgaris genomic DNA segment has 14
other features predicted by gene finders trained with Arabidopsis. Whereas seven ORFs
produce predicted proteins with probable functions that can be deduced from
BLAST analysis, seven other ORFs had predicted protein products without any known
function.It is also interesting to note that anHSF gene is located just 2 Kb downstream of NPR1 in sugarbeet, and the predicted product of this gene is a
DNA-binding HSF protein similar to that specified by the HSFA9 gene that controls early leaf morphogenesis in sunflower [24].We conclude that conserved
microsynteny ofNPR1, CaMP, and CK1PK in three eudicot species suggests strong positive natural selection for the maintenance of
physical linkage of these particular genes whose vital protein products either control
specific nuclear events or are involved in signal transduction.



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*Source: 391259-2008-10-30.xml*