ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2012 Vol. 55, Št. 1: 45-48
Lack of introns in putative parasitism factor gene, expansin (expB2) from pale potato cyst nematode Globodera pallida
Odsotnost intronov v genu za ekspanzin (expB2), verjetnem parazitskem dejavniku, pri beli krompirjevi ogorčici Globodera pallida
Barbara Gerič Stare*, Saša Širca, Gregor Urek
Agricultural Institute of Slovenia, Plant Protection Department, Hacquetova ulica 17, 1001 Ljubljana, Slovenia. Correspondence: barbara.geric@kis.si
Abstract: Expansins are a group of plant cell wall loosening proteins. In animals, functional expansin (EXPB1) has been discovered in the golden potato cyst nematode Globodera rostochiensis. In plant-parasitic nematodes expansins act as the parasitism factors or effectors. Molecular variability of another expansin (expB2) gene was evaluated in the diverse populations of the G. rostochiensis. Comparison of the expB2 gene structure in the two potato cyst nematode species, G. rostochiensis and G. pallida, revealed lack of all four introns in expB2 gene of G. pallida species. Possible loss of introns in Gp-expB2 is discussed.
Keywords: Cell wall degradation, Globodera pallida, effectors, expansin, intron, parasitism factor, plant-parasitic nematode, potato cyst nematode.
Izvleček: Ekspanzini so skupina proteinov, ki zrahlja rastlinsko celično steno. Pri živalih so odkrili funkcionalni ekspanzin (EXP1) pri vrsti rumena krompirjeva ogorčica, Globodera rostochiensis. Pri rastlinsko parazitskih ogorčicah ekspanzini delujejo kot parazitski dejavniki oz. efektorji. Pri G. rostochiensis je bila ovrednotena molekulska raznolikost dodatnega ekspanzinskega gena (expB2). Primerjava strukture gena expB2 pri dveh vrstah krompirjevih ogorčic, G. rostochiensis in G. pallida, je razkrila odsotnost vseh štirih intronov pri vrsti G. pallida v primerjavi z vrsto G. rostochiensis. Predvidevamo možnost izgube intronov pri Gp-expB2.
Ključne besede: razgradnja celične stene, Globodera pallida, efektorji, ekspanzin, intron, parazitski dejavnik, rastlinsko parazitske ogorčice
Introduction
Expansins are a group of proteins that operate by loosening non-covalent interactions between components of the plant cell wall making the individual components vulnerable to attack by other cell wall degrading enzymes (Cosgrove et al. 2000). These proteins were thought to be specific
to plants; however an active expansin EXPB1 has unexpectedly been identified in the plant-parasitic nematode, golden potato cyst nematode Globodera rostochiensis (Woll.) Behrens (Qin et al. 2004). The potato cyst nematodes (PCN) G. rostochiensis and G. pallida (Stone) Behrens are plant-parasitic nematodes which parasitize different Solanaceous plant species. PCN pose a serious threat to potato
production worldwide, and they are subject to strict quarantine regulations in many countries. In Slovenia G. rostochiensis has spread in the last decade (Širca et al. 2010), while G. pallida has been first detected in Slovenian soil just recently (Širca et al. 2012).
When PCN invade a plant, they produce a mixture of lytic enzymes and expansins in their oesophageal glands and secrete them through the stylet into the plant. These proteins assist in the migration of infective juveniles through the host plant's tissues, and in the feeding site formation. Additionally, the host plant's own expansin genes are up-regulated upon nematode infection (Fudali et al. 2008). Expansins B1 and B2 were determined in the EST analysis of G. rostochiensis, while only expansin B1 was found in the G. pallida EST database (Popeijus et al. 2000, http://www. nematodes.org/nembase4/overview.shtml). Molecular variability of expB2 gene was evaluated in the diverse populations of the G. rostochiensis (Gerič Stare et al. 2012).
The aim of this study was to check for the possible presence of the expB2 in G. pallida and to determine its structure.
Materials and methods
DNA was extracted from 10 cysts of the G. pallida population. DNA extraction, amplification of expB2 gene, cloning of the amplicon, isolation of pDNA, sequencing and sequence analysis were performed as previously described in detail by Gerič Stare et al. (2012) for the orthologous expB2 gene in G. rostochiensis (Gr-expB2). Primers were designed based on the Gr-expB2 mRNA sequence (AJ311902) coding for the putative functional expansin.
Results
The primer set designed for the expB2 gene of G. rostochiensis yielded a much shorter PCR amplicon with the G. pallida genomic DNA (543 bp in G. pallida vs. 1.129 - 1.153 bp in G. rostochiensis). Sequence analysis revealed high homology to previously determined Gr-expB2 precursor (AJ311902) by BLASTN with E value
0.0. Alignment of this G. pallida sequence with the previously determined genomic sequences of Gr-expB2 (FJ705444, GQ152151 - GQ152166, GQ152168 - GQ152288) revealed lack of all four introns. Due to the high homology of the coding region of the Gr-expB2 gene, this sequence was designated Gp-expB2. Gp-expB2 shared 99.6% identity with Gr-expB2 cDNA (GQ152150). Further, no highly homologous sequence to Gp-expB2 could be found in the G. pallida genome sequence (http://www.sanger.ac.uk/resources/downloads/ helminths/globodera-pallida.html), although there are several sequences similar to expansin, except for including introns. The sequences Gp-expB2 reported here was deposited at Genbank with the accession number GQ152167.
Discussion
In the database of G. pallida EST sequences (http://www.nematodes.org/nembase4/index. shtml) there are no expansin B2 related sequences. Nonetheless, we have determined orthologous sequence using the primer set developed for Gr-expB2 in a PCR with G. pallida gDNA. Gp-expB2 sequence showed high similarity to exons of determined Gr-expB2 sequences but lack of all four introns found in G. rostochiensis. Closely related species usually possess conserved introns, but there are also examples where introns are present in one species but not in closely related one (Kent and Zahler, 2000). From an alignment of two sequences it is not possible to tell whether the introns are being lost or gained.
One hypothesis is that introns may be lost during repair of double-stranded breaks in DNA helix in a repair mechanism involving reverse transcription of mRNA and reintegration of the synthesized cDNA into the genome by homologous recombination. Another example of a gene where this process might have happened is a gene for ß-1,4-endoglucanases in Heterodera glycines (Hg-eng5), a parasitic factors of plan-parasitic nematodes that contains no introns (Gao et al. 2004, Kyndt at al. 2008). On the other hand, a recent horizontal gene transfer event from a prokaryote was suggested for lack of introns in Hg-eng5 (Gao et al. 2004). Furthermore, formation of genes without intron from paralogous genes with introns
in eukaryotes might arise from gene duplication process involving reverse transcription of mRNAs with recombination of the synthesized cDNA in the genome (Boudet et al. 2001).
Conclusion
Comparison of orthologous expB2 gene in potato cyst nematodes revealed lack of all four introns in G. pallida compared to G. rostochiensis.
Povzetek
Ekspanzini so proteini, ki z zrahljanjem neko-valentnih vezi pomagajo pri razgradnji rastlinske celične stene. Pri živalih so odkrili funkcionalni ekspanzin (EXP1) pri vrsti rumene krompirjeve ogorčice Globodera rostochiensis (Nematoda). Pri rastlinsko parazitskih ogorčicah ekspanzini delujejo kot parazitski dejavniki oz. efektorji. Pri G. rostochiensis je bila ovrednotena molekulska raznolikost dodatnega ekspanzinskega gena (expB2). Z uporabo začetnih oligonukleotidov predhodno razvitih za Gr-expB2 smo določili
prisotnost gena expB2 tudi pri sorodni vrsti bele krompirjeve ogorčice G. pallida. Primerjava strukture gena expB2 je razkrila odsotnost vseh štirih intronov pri Gp-expB2 v primerjavi Gr-expB2. Do izgube intronov pri Gp-expB2 bi lahko prišlo na različne načine. Prvi je mehanizem popravljanja poškodb dvoverižne DNA, ki vključuje obratno prepisovanje mRNA v cDNA in reintegracijo le te v genom s homologno rekombinacijo. Drug možen mehanizem je nedaven horizontalni prenos gena iz prokariontov. Tretja možnost nastanka genov brez intronov pri evkariontih je z podvojitvijo paralognih genov z introni v procesu, ki vključuje obratno prepisovanje mRNA in rekombinacijo tako nastale cDNA v genom.
Acknowledgements
This work was supported by the Slovenian Research Agency and the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (V4-0324 and BI-FR/07-08-INRA-003). This work benefited from links funded via COST 872 action.
References
Boudet, N., Aubourg, S., Toffano-Nioche, C., Kreis, M., Lecharny, A., 2001. Evolution of intron/ exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila. Genome Research, 11, 2101-2114.
Cosgrove, D.J., 2000. Loosening of plant cell walls by expansins. Nature, 407, 321-326.
Fudali, S., Sobczak, M., Janakowski, S., Griesser, M., Grundler F.M.W., Golinowski. W., 2008. Expansins are among plant cell wall modifying agents specifically expressed during development of nematode-induced syncytia. Plant Signaling & Behavior, 3 (11), 969-971.
Gao, B.L., Allen, K.D., Davis, E.L., Baum, T. J., Hussey, R.S., 2004. Developmental expression and biochemical properties of a beta-1,4-endoglucanase family in the soybean cyst nematode, Hete-rodera glycines. Molecular Plant Pathology, 5, 93-104.
Gerič Stare, B., Lamovšek, J., Širca, S., Urek, G., 2012 Assessment of sequence variability in putative parasitism factor, expansin (expB2) from diverse populations of potato cyst nematode Globodera rostochiensis. Physiological and Molecular Plant Pathology, in press (doi:10.1016/j. pmpp.2012.04.002)
Kent, W.J., Zahler, A.M., 2000. Conservation, regulation, synteny, and introns in a large-scale C. briggsae-C. elegans genomic alignment. Genome Research, 10, 1115-1125.
Kyndt, T., Haegeman, A., Gheysen, G., 2008. Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event. BMC Evolutionary Biology, 8, 305.
Popeijus, H., Blok, V.C., Cardle, L., Bakker, E., Phillips, M.S., Helder, J., Smant, G., Jones, J.T., 2000 Analysis of genes expressed in second stage juveniles of the potato cyst nematodes Globodera rostochiensis and G. pallida using the expressed sequence tag approach. Nematology, 2000, 2 (5), 567-574.
Qin, L., Kudla, U., Roze E.H.A., Goverse, A., Popeijus, H., Nieuwland, J., Overmars, H., Jones, J.T., Schots, A., Smant, G., Bakker, J., Helder, J., 2004. A nematode expansin acting on plants, Nature, 427, 30.
Širca, S., Gerič Stare, B., Strajnar, P., Urek, G., 2010, PCR-RFLP diagnostic method for identifying Globodera species in Slovenia. Phytopathologia Mediterranea, 49, 361-369.
Širca, S., Gerič Stare, B., Strajnar, P., Urek, G., Miklič Lautar, I., 2012. First report of the pale potato cyst nematode Globodera pallida from Slovenia. Plant disease, 96, 773.
http://www.nematodes.org/nembase4/overview.shtml