Two LTR retrotransposon elements within the abscisic acid gene cluster in Botrytis cinerea B05.10, but not in SAS56 Ming Zhao Jin yan Zhou You jiu Tan Wei wei Song Zhi dong Li Hong Tan* *Corresponding author Financial support: Key Project of Chinese Academy of Sciences (No.KSCX2-YW-G016) and the Hi-Tech Research and Development Program (863) of China (No.2006AA10A213). Keywords: Botrytis cinerea, genomic sequence analysis, in silico, LTR retrotransposon.
The plant hormone abscisic acid has huge economic potential and can be applied in agriculture and forestry for it is considered to be involved in plant resistance to stresses such as cold, heat, salinity, drought, pathogens and wounding. Now overproducing strains of Botrytis cinerea are used for biotechnological production of abscisic acid. An LTR retrotransposon, Boty-aba, and a solo LTR were identified by in silico genomic sequence analysis, and both were detected within the abscisic acid gene cluster in B. cinerea B05.10, but not in B. cinerea SAS56. Boty-aba contains a pair of LTRs and two internal genes. The LTRs and the first gene have features characteristic of Ty3/gypsy LTR retrotransposons. The second gene is a novel gene, named brtn, which encodes for a protein (named BRTN) without putative conserved domains. The impressive divergence in structure of the abscisic acid gene clusters putatively gives new clues to investigate the divergence in the abscisic acid production yields of different B. cinerea strains.
The phytopathogenic ascomycete Botrytis cinerea, is known to produce the plant hormone abscisic acid (ABA), which plays a major role in several steps of plant growth and development, such as stomatal closure, embryo and seed dormancy, seed germination and the adaptation to environmental stress (Tudzynski and Sharon, 2002). ABA has huge economic potential and can be applied in agriculture and forestry for it is considered to be involved in plant resistance to stresses such as cold, heat, salinity, drought, pathogens and wounding (Tudzynski and Sharon, 2002). The production yields reported of B. cinerea ABA are 0 ~ 1.6 g/L in axenic culture (Wu and Zheng, 1997; Tan and Li, 1998; Wu and Shi, 1998; Tudzynski and Sharon, 2002; Liang et al. 2004). Now overproducing strains of B. cinerea are used for biotechnological production of ABA (Tan and Li, 1998). Recently, an ABA gene cluster that contains gene bcaba1, bcaba2, bcaba3, and bcaba4 in B. cinerea SAS56 was identified (Siewers et al. 2004; Siewers et al. 2006). The Whole Genome Shotgun (WGS) sequences of B. cinerea B05.10 are public available at http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=fungi. In this work, the putative ABA gene cluster of B. cinerea B05.10 was identified by an in silico genomic sequence analysis. To our surprise an impressive divergence in structure of the two ABA gene clusters was identified. An LTR retrotransposon and a solo LTR were detected within the ABA gene cluster in B. cinerea B05.10, but not in B. cinerea SAS56. For it has been shown that retrotransposons may contribute to the expression pattern of many host genes (Kashkush et al. 2003), this divergence in structure of the two ABA gene clusters putatively gives new clues to survey the divergence in the ABA production yields of different B. cinerea strains. Nucleotide sequences of bcaba1(AJ609392), bcaba2 (AJ851088), bcaba3 (AM237449) and bcaba4 (AM237450) were obtained from GenBank and used to query the whole genome sequence (WGS) of B. cinerea B05.10 by BLASTN search provided by the Broad Institute (Altschul et al. 1997). The annotated features of the genome sequences from the bcaba4 gene to the bcaba3 gene were viewed using Browse Region provided by the Broad Institute. The scheme of the putative ABA gene cluster of B. cinerea B05.10 was drawn by the computer software Chem Draw ultra 8.0. The DNA sequence of B. cinerea B05.10 from the bcaba4 gene to the bcaba3 gene was extracted using Browse Region provided by the Broad Institute. The repetitive elements were analyzed in RepeatMasker. The termini of the LTRs were confirmed by manual inspection. Protein domains were identified using RPSBLAST (Marchler-Bauer and Bryant, 2004). Sequences of Saccharomyces cerevisiae tRNAs used for identification of PBSs were obtained from the Genomic tRNA Database (http://lowelab.ucsc.edu/GtRNAdb/Scere) (Lowe and Eddy, 1997). The polypurine tract (PPT) and the target-site duplications (TSD) of LTR retrotransposon were investigated by manual inspection. The putative ABA gene cluster of B. cinerea B05.10 is located in the supercontig 41 from 72143 to 91420 bp (in the cont1.1706, NZ_AAID01001706). Sequence identity analysis reveals that each ABA gene of strain B05.10 shows a high degree of similarity to the ABA gene of strain SAS56 (gene bcaba1, bcaba2 and bcaba4 with 99% identity, respectively; gene bcaba3 with 98% identity). It is noteworthy that an astonishing divergence in structures of the two ABA gene clusters was identified. In B. cinerea SAS56, gene bcaba3 is located 3.7 kb upstream of gene bcaba1 (Figure 1a), while in B. cinerea B05.10, gene bcaba3 is located about 10.5 kb upstream of gene bcaba1 and two putative genes were detected within this region. A sequence similarity search by BLASTP and BLASTN revealed that the first gene is an LTR retrotransposon gag-pol gene, and the second is a novel gene (Figure 1b). To investigate the structure features of ABA gene cluster of B. cinerea B05.10, about 20 kb of WGS sequences of B. cinerea B05.10 from gene bcaba4 to gene bcaba3 was extracted. The extract was analyzed with RepeatMasker. A solo LTR and a LTR retrotransposon that was designated as Boty-aba were identified. Boty-aba is
flanked by 5 bp direct repeats (CATTC) representing target-site
duplications (TSD). It contains 6603bp with a pair of LTRs flanking
and two internal deduced genes. The LTRs contain 561bp (left) and
570bp (right), respectively. And they share 93.8% identity. A sequence
similarity search by BLASTN demonstrates that both left and right
LTR show significant similarity to the LTR of Boty (X81790),
with 80.2% and 82.0% identity, respectively. Structure factures
analysis reveals that the three LTRs contain the 5’-terminal
and 3’-terminal sequences (5’TG...CA3’) and perfect
short inverted terminal repeats of 7 bp (TGTTACG…CGTAACA)
(Diolez et al. 1995). The presumed TATA boxes
are found in the three LTRs. Boty-aba and Boty contain
the identical primer binding sites (PBSs) for first-strand reverse
transcription with 9 nt (5’-TTTGAGCAC-3’) immediately
downstream of the left LTR. They both use self-priming mechanism
to initiate synthesis of reverse transcripts (Lin
and Levin, 1997). The polypurine-rich sequence that corresponds
to the primer binding site for plus-strand DNA (PPT) synthesis
is located immediately upstream of the right LTR, and the sequence
of PPT in Boty-aba is 5’-AGGCTAAGAAGGGGATAG-3’.
The solo LTR is located 448 bp upstream of Boty-aba and
flanked by 5 bp direct repeats (CTCAT) representing TSD; it is
inverted and is identical with the left-LTR of Boty-aba.
The first internal gene of LTR retrotransposon is a gag-pol gene. This gene has the same transcription direction as gene bcaba3 and contains 4255 nucleotides with 3 exons coding for 1302 amino acids. Conserved domain search analysis by RPSBLAST demonstrates that this gene encodes a polyprotein with a retrotrans_gag (gag) domain, a C2HC zinc fingers, a reverse transcriptase (RT) domain, and an integrase core (IN) domain (Figure 1c). This polyprotein does not contain protease and RNase H domains. The analysis of the conserved domains by RPSBLAST is shown in Figure 2. The fact that the domains of Boty-aba within the pol gene are arranged in the order RT and IN reveals that Boty-aba belongs to the Ty3/gypsy group of retrotransposons. The fact that the LTRs of Boty-aba are not identical and there are no protease and RNase H domains suggests that Boty-aba is an ancient element and it possibly cannot retrotranspose. The second internal gene of LTR retrotransposon is 597 bp long and encodes 199 amino acids. It is transcribed divergently. No putative conserved domain has been detected using RPSBLAST. On the basis of these results, we believe that this is a novel gene, which we designate brtn (B. cinerea retrotransposon novel) and the deduced protein is named BTRN. The structural features of Boty-aba are depicted in Figure 1b. We also identified an EST (W0AA017ZF01C1) shows high sequence similarity to gene brtn (100% identities). We presume that the internal promoter of the flanking LTRs drive the transcription of gene brtn. Retrotransposons are a widespread and important class of eukaryotic mobile genetic elements that have a central role in the structure, evolution, and function of eukaryotic genomes (Bennetzen, 2000 ; Kidwell and Lisch, 2001). Recent reports have shown that retrotransposons contribute to the formation of genome structure and to the expression pattern of many host genes (Kashkush et al. 2003). For example, when Wis 2-1A retrotransposons are activated in wheat, the expression of several adjacent genes is activated or silenced by producing sense or antisense transcripts of those genes (Kashkush et al. 2003). Matsubara concluded that at least 3 transposable elements in Hf1 gene that plays a key role in the expression of floral colour in petunias govern anthocyanin biosynthesis of commercial petunias (Matsubara et al. 2005). It is not known whether Boty-aba has an affect on the expression of ABA genes. This divergence in structure of the ABA gene clusters gives new clues to survey the divergence in the ABA production yields of different B. cinerea strains. ALTSCHUL, S.F.; MADDEN, T.L.; SCHÄFFER, A.A.; ZHANG, J.H.; ZHANG, Z.; MILLER, W. and LIPMAN, D.J. 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