# A Nest of LTR Retrotransposons Adjacent the Disease Resistance-Priming GeneNPR1 in Beta vulgaris L. U.S.  Hybrid H20

Authors: David Kuykendall; Jonathan Shao; Kenneth Trimmer
Journal: International Journal of Plant Genomics
 (2009)

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## Abstract

A nest of long terminal repeat (LTR) retrotransposons (RTRs), discovered by LTR_STRUC analysis, is near core genes encoding the NPR1 disease resistance-activating factor and a
heat-shock-factor-(HSF-) like protein in sugarbeet hybrid US H20.SCHULTE, a 10 833 bp LTR retrotransposon, with 1372 bp LTRs that are 0.7% divergent, has two ORFs with unexpected introns but encoding a reverse transcriptase with rve and Rvt2 domains similar to Ty1/copia-type retrotransposons and a hypothetical protein. SCHULTE produced significant nucleotide BLAST alignments with repeat DNA elements from all four families of plants represented in the TIGR plant repeat database (PRD); the best nucleotide sequence alignment was to ToRTL1 in Lycopersicon esculentum. A second sugarbeet LTR retrotransposon, SCHMIDT, 11 565 bp in length, has 2561 bp LTRs that share 100% identity with each other and share 98-99% nucleotide sequence identity over 10% of their length with DRVs, a family of highly repetitive, relatively small DNA sequences that are widely dispersed over the sugarbeet genome. SCHMIDT encodes a complete gypsy-like polyprotein in a single ORF. Analysis using LTR_STRUC of an in silico deletion of both of the above two LTR retrotransposons found that SCHULTE and SCHMIDT had inserted within an older LTR retrotransposon, resulting in a nest that is only about 10 Kb upstream of NPR1 in sugarbeet hybrid US H20.


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## Body

## 1.  Introduction

Retrotransposons are now recognized as movers and shapers of plant genome evolution (see reviews
[1, 2]). That retrotransposon elements account for much of the sugarbeet (Beta vulgaris L.) genome was shown by
the identification [3] of repetitive DNA sequences in Beta vulgaris similar to long interspersed nuclear elements
(LINEs), a type of retrotransposon without long terminal
repeats (LTRs), and other
repetitive DNA sequences that resembled LTR retrotransposons of the Ty1-copia class. A repeated DNA sequence
in Beta procumbens was described as 		“Athila-like” [4] since it was deduced to
be part of a long terminal repeat with similarity to the Athila retrotransposon from Arabidopsis.Prior to the present study, pDRV sequences [5]
were known simply as a family of short highly amplified DNA repeats shown by
fluorescent in situ hybridization
(FISH) technique to be widely dispersed over all 18 chromosomes of sugarbeet.Vulmar1, a mariner-class DNA transposon in Beta
vulgaris [6], is 3909 bp, has 32 bp terminal inverted repeats, and
encodes, in a single ORF, a transposase with a characteristic “DDE” signature
motif.  Polymerase chain reaction (PCR)
and fluorescent in situ hybridization (FISH) were used [6] to identify and to
establish an abundance of En/Spm-like
transposons in sugarbeet.Coe1, a DNA transposon within apparent
LTRs and other retrotransposon-like features, was discovered on a sugarbeet
genomic BAC carrying the NPR1 disease
resistance-priming gene [7–9]. This recent discovery in Beta vulgaris of a unique 16.3 Kb CACTA En/Spm-like transposon named Coe1 [7] was followed by the finding of
conserved microsynteny of NPR1 with
another core plant gene whose predicted product has high similarity to a
DNA-binding HSF protein [8].  About 70 Kb
of repetitive DNA separates the HSF gene and NPR1 from another small core
gene cluster with a CaMP gene
specifying a signal peptide calmodulin-binding protein and a gene encoding a
CK1-class protein kinase gene [8], greatly extending and disambiguating the
results of the initial sequencing and partial in silico analysis of an NPR1 gene-carrying sugarbeet BAC [9]. In summary, our laboratory has identified,
sequenced, and annotated a bacterial artificial chromosome (BAC) carrying the NPR1 disease resistance priming gene of
sugarbeet, Beta vulgaris L. [7–9].Class I
transposable elements which use reverse transcriptase to transpose via an RNA
intermediate are termed retrotransposons. In order to identify possible LTR
retrotransposons with LTRs, an intergenic region of
repetitive DNA was examined by LTR_STRUC analysis, and this report details the discovery of a nest of retrotransposons about 10 Kb upstream from theNPR1 disease resistance gene in sugarbeet H20. This nest appears to have formed when both a copia-type and a gypsy-type elements inserted within an older LTR retrotransposon.
Two full-length sugarbeet LTR retrotransposons are described herein for the
first time.


## 2. Materials and Methods

Identification
of a sugarbeet BAC carrying theNPR1 disease resistance control gene was described [9]. Genbank accession DQ851167
represents a partial sequence; the 38.6 Kb segment was the largest contig at
that time. Subsequently the entire 130 Kb contiguous fragment was sequenced and
annotated (Genbank accession EF101866). Basic methods used for DNA sequence
analysis were described [9], and construction of the BAC library was detailed
[10]. In the present study, LTR analyses of the NPR1 BAC were performed
using LTR_STRUC
[11], and LTR Finder
[12]. Programs, einverted
[13] (http://bioweb.pasteur.fr/seqanal/interfaces/einverted),
and EMBOSS (http://emboss.sourceforge.net/) [13] were used to
identify inverted repeats, and repeats were also found using NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).
An EST database for sugarbeet (http://genomics.msu.edu/sugarbeet/blast.html)
was employed for both nucleotide and protein BLAST to explore possible
functional gene expression [14]. Subsequent analysis of DNA sequence data was
performed using Lasergene version 6 (DNASTAR, Madison, Wis, USA).
BLAST was used to identify the most similar protein products of LTR
retrotransposons in other plant species. Multiple alignments were performed
using MegAlign from the DNASTAR suite. Neighbor joining tree, or cluster
analysis, was performed using MEGA 4 software (http://www.megasoftware.net/).


## 3. Results and Discussion

A genomicNPR1 disease resistance priming gene-carrying BAC [7–9] was
subjected to LTR_STRUC and LTR FINDER analyses, and two distinct full-length LTR
retrotransposons were identified (Figure 1). Depicted are RTR1 and RTR2, two LTR
retrotransposons that we also term SCHULTE and SCHMIDT, respectively, as well as a previously described element, Coe1, a DNA transposase gene within apparent LTRs and other
retrotransposon-like features [7]. These repetitive DNA elements are
intergenic, between two small clusters of core genes: HSF and NPR1 genes
separated from CaMP and CK1PK genes encoding a signal peptide
calmodulin-binding protein and a “casein kinase 1-class protein kinase,”
respectively.Figure 1
BAC physical
map showing two LTR retrotransposonsSCHULTE and SCHMIDT both highlighted in green
and with darker green LTRs with rounded corners. On the other hand, the
previously reported CACTA transposon Coe1,
highlighted in light beige, has short tan LTRs, a yellow DNA transposase gene
and orange ORF1 and ORF2. Exons are rectangles, and direction of arrows
indicates direction of transcription.  Exons
of core plant genes are blue, and exons of hypothetical protein genes are red. Solid
lines between exons depict introns. Scale is in 2 Kb increments.  BAC is about 130 kilobase pairs. SCHULTE has an integrase gene in orange and a hypothetical gene in red. SCHMIDT has a single ORF retroelement reverse transcriptase polyprotein gene in orange.SCHULTE, a 10 833 bp long LTR
retrotransposon, has 1372 bp LTRs sharing only 99.3% nucleotide sequence
identity. The 0.7% divergence in the LTRs
of SCHULTE indicates about ten base
substitutions occurred since insertion/transposition. This old, somewhat degraded retrotransposon has two ORFs encoding a Ty1/copia-like integrase/reverse transcriptase and a hypothetical protein
(Figure 2). Unexpected introns, uncharacteristic of retrotransposon genes, may be
the result of frameshifts and point mutations. SCHULTE has 98% nucleotide sequence identity over ≥9 Kb
with a 9.7 Kb DNA fragment (DQ374026) and 1.3 Kb of a 5.3 Kb DNA fragment
(DQ374025), each fragment of BAC62 [14]. BAC62 carries a Beta vulgaris L. genomic region adjacent a Beta procumbens translocation carrying a nematode resistance gene
[15], thus BAC62 has a SCHULTE-like
retrotransposon.Figure 2
A detailed schematic ofBeta vulgaris retrotransposons SCHULTE and SCHMIDT in red. Various numbered inverted repeat (IR) sequences in either
light or dark blue or violet have arrows indicating relative direction.  Green lines show the location of the DNA
sequence motif CACTATAA.  Heavily dotted
ovular regions depict the size and position of the LTRs.  The lightly dotted region shows the size of
the whole retroelement.  A green box
shows the location of the polymerase binding site.  Boxes show the position and size of
exons.  Lines between exons indicate
introns.  A centrally located small
yellow box depicts the active site of the retroelement. An orange box shows the
location of the polypurine tract.  Scale
is in Kb and is located underneath the illustrations in increments of 2 Kb per
tick.  Names of putative genes are
located above the illustrations.Named to honor an
author of the first-described physical map of the afore-mentioned region,SCHULTE is the first full-length
retrotransposon sequence from Beta
vulgaris to be reported. Since two out of the three B. vulgaris BACs sequenced to date, BAC62 and the NPR1-carrying BAC,   carry a SCHULTE-like element, there are likely a
very large number of SCHULTE-like LTR
retrotransposons in the sugarbeet genome. However, FLC, or the flowering control gene-carrying BAC [16], did not carry
a SCHULTE-like element.SMART analysis
showed that the predicted product of theSCHULTE reverse transcriptase gene has rve and Rvt2 protein domains.  An alignment by MegAlign of the conserved rve and Rvt2 (Figure 3) domains of similar Ty1-copia-like plant retrotransposon-encoded proteins, identified
by BLAST, were analyzed by neighbor joining in MEGA 4 to assess structural
relatedness (Figure 4). As shown in Figure 3, the predicted product of the Beta vulgarisSCHULTE reverse transcriptase gene has conserved rve and Rvt2 domains shared among highly similar domains of products of LTR
retrotransposons from Medicago truncatula, Vitis vinifera,Oryza sativa japonica, Zea
mays, and Glycine max.  Except for the Solanum demissum and Vitis
vinifera accessions, the Rvt2 domain evidently has a conserved YVDDIIF active site (Figure 3).Figure 3
Amino acid residue alignment ofSCHULTE’s integrase (top) and reverse
transcriptase (bottom) domains.  The
active site of the RT domain from Beta
vulgaris is boxed in along with corresponding sections of other reverse
transcriptase proteins from different plants.
Amino acids matching the consensus sequence are shaded. Numbers indicate
cumulative amino acid positions; Arabidopsis thaliana (AAG50698), Beta vulgaris (ABM55238), Glycine max (AAO73521), Medicago truncatula (ABD32582), Oryza sativa japonica (AAR87335), Phaseolus vulgaris (AAR13298), Solanum demissum (AAT38797), Vitis vinifera (CAN74951), Zea mays (ABF67921).Figure 4
Similarity tree constructed by neighbor
joining method of the reverse transcriptase domain ofSCHULTE from Beta vulgaris along with corresponding domains of other reverse transcriptase proteins from
different plants.  The numbers on the
branches are bootstrap (confidence) values.
Genbank accession numbers of amino acid sequences are given following
plant names.Similar LTR
retrotransposon gene products inArabidopsis
thaliana, Solanum demissum, and
particularly in Phaseolus vulgaris are structurally divergent (Figures 3 and 4). A search of the TIGR plant repeat
database revealed that SCHULTE produced nucleotide sequence matches with many different copia-like retrotransposons in all four families: Brassicaceae,
Fabaceae, Gramineae, and Solanaceae. The best
PRD nucleotide sequence alignment match (E=3.8e−232) was to ToRTL1 in Lycopersicon esculentum.Probable
expression of integrase/reverse transcriptase gene(s) in activeSCHULTE-like retrotransposon was shown
by BLAST alignment (E=0.0) of the ORF with BI643218, an EST, or expressed sequence
tag. Expression of both LTRs was clearly evidenced by alignments, E=0.0, with
ESTs BI698297 and BI698341. Four other ESTs showing some alignment (E=e−151 to E=9e−36) also suggest other likely active SCHULTE-like elements.Another LTR retrotransposon discovered
using LTR_STRUC and LTR FINDER,SCHMIDT was so named to honor a pioneering researcher of repeat DNA elements in Beta. SCHMIDT,  11 565 bp retroelement,
encodes a complete Ty3-gypsy-class
polyprotein in a single ORF without introns. The SCHMIDT reverse transcriptase gene has all of the domains expected of an intact retroelement polyprotein,
and the domain order is indicative of Ty3-gypsy-class. SCHMIDT has 2561 bp LTR sequences
with 100% identity, consistent with this transposable element still being
active.EMBOSS analysis of
the 130 KbNPR1-carrying sugarbeet
BAC revealed the presence of at least 24 inverted repeat (IR) sequences but,
for the purposes of this report, let us describe only those inverted sequences
associated with LTR retrotransposons SCHULTE and SCHMIDT. The following two pairs of IR 8/21 inverted repeat sequences are
associated with SCHULTE (Figure 2).
The IR 8/21 inverted repeat sequences
share 94% identity (18/19). 35690cttagtttgtacctttgtt35708||||||||||||||||||35781gaatcaaacaaggaaacaa3576326302aacaaaggaacaaactaag26320||||||||||||||||||35708ttgtttccatgtttgattc35690 The following pair of IR 22 inverted sequences, associated
with SCHULTE (Figure 2), are 9.5 Kb
apart and share 80% identity (shown below), but this pair of IR 22 are also direct repeats with 96%
identity. 26751->26786aaaaagaaaatctgttttggaaaagattttattttt|||||||||||||||||||||||||||||tttttattttagaaaagattttgtctaaaagaaaaa36247<-36212SCHMIDT has a pair of inverted repeat IR 9 sequences upstream of the single reverse transcriptase
polyprotein gene and just downstream of polymerase binding site (Figure 2).
These IR 9 inverted repeat sequences
share 100% identity (23/23) and contain only nucleotides A and T.
46146aaaatttttttttttttttttta46168|||||||||||||||||||||||46331ttttaaaaaaaaaaaaaaaaaat46309Over about
one-tenth of their length, each LTR sequence ofSCHMIDT shares 97% nucleic acid sequence identity with pDRV, a family of Beta vulgaris short repeated DNA sequences known as rich in DraI (TTTAAA) restriction enzyme
recognition sites [5].  For example,
eight DraI sites are carried by pDRV1, a 434 bp repeated sequence [5]. Each LTR of SCHMIDT has 10 DraI sites,
and the full-length SCHMIDT retrotransposon
has twenty-four DraI (TTTAAA) sites. Perhaps pDVR conserved sequences were originally
a preferred recognition site for insertion; they seem to have evolved into an
integral part of the LTRs of SCHMIDT-like
retrotransposons. In any case, the observed high degree of nucleotide sequence
identity that the LTRs of SCHMIDT have
with pDRVs, highly reiterated
sequences rich in nucleotides A and T [5], is very interesting since pDRV repeat sequences, originally
visualized by FISH, are dispersed over all 18 sugarbeet chromosomes  [5].Figure5 shows
results of alignment by MegAlign of
conserved rve and Rvt1 domains of the protein products
from SCHMIDT-like plant
retrotransposons identified by BLASTp from various plant species, and Figure 6
shows a neighbor joining analysis tree by MEGA 4 showing structural
relatedness. A highly conserved FIDDILI active site in the Rvt1 domain is noted in particular (Figure 5).  The predicted SCHMIDT reverse transcriptase polyprotein gene product shows high
structural similarity with a conserved region of proteins encoded by similar
LTR retrotransposons from Cicer
arietinum, Medicago truncatula, Oryza sativa japonica, Sorghum bicolor, and Zea mays AAL59229. Somewhat similar
LTR retrotransposons from Glycine max, Primula vulgaris, Vitis vinifera, Solanum
demissum, Hordeum vulgare, and Zea mays AAM94350 produce structurally divergent products (Figure 6). Against the
TIGR plant repeat database, SCHMIDT produced good, E≤e−50, BLAST nucleotide sequence
identity which matches primarily with Prem1-
and Xilon1-like gypsy-like RTRs in Zea, Oryza, Sorghum, and Triticum. This finding suggests divergent evolution, where a SCHMIDT-like ancestor originated in monocots, then, upon
lateral transfer to sugarbeet, natural selection for structural similarity or convergence
in a new genetic background resulted in a high degree of amino acid similarity
of the protein product with other gypsy-like
retrotransposons in eudicots. The predictions of convergent evolution are structurally
similar proteins encoded by phylogenetically distinct retrotransposons. Whether
similar sequences arose through convergent or divergent evolution, it is
interesting to simply note that SCHMIDT has a significant degree of nucleotide sequence identity primarily with certain gypsy-like retrotransposons found in
monocots.Figure 5
Amino acid residue alignment of the reverse
transcriptase (top) and integrase (bottom) domains ofSCHMIDT from Beta vulgaris,
with its active site boxed in, along with corresponding sections of other
reverse transcriptase proteins from different plants.  Amino acids matching the consensus sequence
are shaded. Numbers indicate cumulative amino acid positions; Arabidopsis
thaliana (BAB10790), Asparagus
officinalis (ABD63142), Beta vulgaris (ABM55240), Cicer arietinum (CAC44142), Glycine max (AAO23078), Hordeum vulgare (AAK94517), Medicago truncatula (ABO81153), Oryza sativa japonica (ABA92233), Primula vulgaris (ABD78322), Solanum demissum (AAW28577), Sorghum bicolor (AAD22153), Vitis vinifera (CAN79321), Zea mays (AAM94350), Zea mays (AAL59229).Figure 6
Similarity tree constructed by neighbor
joining method of the reverse transcriptase domain ofSCHMIDT from Beta vulgaris along with corresponding domains of other reverse transcriptase proteins from
different plants.  The numbers on the
branches are bootstrap (confidence) values.
Genbank accession numbers of amino acid sequences are given following
plant names.Expression of retroelements
similar to SCHMIDT in sugarbeets
is suggested by the finding thatSCHMIDT gave BLAST alignments with the following ESTs: BI643170, BI643158, BI698360, and
BI643246 (E=10−161, 3E=10−79, E=10−68, and
5E=10−59, resp.). These four BLAST hits represent only about
0.02% of the ESTs in the collection.An older LTR
retrotransposon, which had been interrupted by subsequent insertions ofSCHULTE and SCHMIDT, became evident (Table 1) when LTR_STRUC analysis was
performed on a sequence having an in silico deletion of the LTR
retrotransposons SCHULTE and SCHMIDT. Although very degraded and unclassifiable,
the older LTR retrotransposon was deduced to be 5395 bp with 780 bp LTRs
sharing 99% identity.Table 1
Several LTR
retrotransposons discovered within a TE nest, in addition toCoe1 [7], by LTR_STRUC analysis of a sugarbeet
genomic BAC carrying NPR1.
FeatureSCHULTESCHMIDTOlder LTR-RTRCoe1 [7]Active siteYVDDIILFIDDILISCDDVLLYVDDIILLength of RTR10 833 bp11 565 bp5395 bp (before two TE insertions)14 531 bpLength of LTR1372 bp2561 bp780 bp169 bp5′ LTR-3′ LTR
Identity (%)99.3%100.0%99.0%96.4%Number of open
reading frames (ORFs)21235′ beginning and 3′
end of flanking region (duplication)ATTTTCGCTCGCTTGCTACTClass & domains presentCopia-like
Integrase and RTase domainsAll domains expected of a completeGypsy-like retrotransposonPutative RT domainsCoe1 [7],   class II within a class I, DNA
transposase/Copia-like RTase pseudogeneIn
conclusion, the relatively small repetitive DNA sequences previously described as
“pDRVs” can now be seen as a part of the
LTRs of SCHMIDT-like retrotransposons.Planned research will address possible effects
of retrotransposons on the expression of core plant genes including theNPR1 disease resistance-priming gene
immediately downstream of the LTR retrotransposon nest.


## 4.  Conclusions

An LTR retrotransposon nest
consisting of an older retroelement into which both agypsy-like SCHMIDT and a copia-like SCHULTE inserted was identified, and properties of the
retrotransposons were described. Since LTR retrotransposons are driving forces
in plant genome evolution (see reviews [1, 2]), they may have tremendous
potential usefulness in genetic manipulation and genome modification to enhance
agricultural profitability and sustainability.



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