GENES & GENOMICS 31 (6) : 443-450 (December 20(9)

CThe Genetics Society of Korea

Structure of Ribosomal RNA Gene and Phylogeny of Nosema Isolates in Korea Jiyoung Choit, Jonggill Kim" Youngcheol Choi\ Sung-Hee Nam', James Russele, Wontae Kim l , Jan WUytS3, Yeonho Je 4 and Gilsang Jeong'· 'Department of Agricu.Itun.l. Biology, National Academy of Agricu.Iturai Science. RDA, Suwon 441-853, Republic of Korea IDepartment of Btology, University of California. Riven;ide 92521. USA iTIepartment of Plant Biology. F1ander Intenmiversity Institute for Biotechnology (VIB), Ghent Univenlity, Teehnologiepark 9T1, 89052 Ghent, Belgium fDepartment of Agricultural Bioteehnology, Seoul National Univenlity. Seou1151-742, Republic of Korea

Received October 19, 2009; accepted November 2, 2009

ABSTRACT The ribosomal RNA (rRNA) gene region of the four Nosema sp. isolates (COl, CO2, C03 and C04) from Pieris rapae in Korea has been examined. Complete DNA sequence data (3779 bp) of the rRNA gene of Nosema sp. COl are presented for the small subunit gene (SSU rRNA: 1236 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene a..sU rRNA: 2506 bp). The seoondary structures of Nosema sp. COl SSU and LSU rRNA genes are oonsb'ucted and described. The SSU rRNA showed a hypervariable V4 region identified four additional stems

including a pseudokrnt. Phylogenetic analysis based on the SSU rRNA - " that the rOOf isolates belong to the 'true' Nosema group. In contrast to the NosemalVairimorpha clade, the members of the group are highly divergent.

Key 'l'r'Ords: ribosomal RNA, RNA seoondary structure, 'true' Nosema group, NosemaIVairimorpha clade.

INTRODUCTION Microsporidia belongs to the phylum Microsporidia. They are composed of roore than 143 genera and 1,200 species that are obligate intracellular parasites of all animal groups, especially insects, fish and mammals (Wittner and Wei"., 1999). Insects of nearly all orders are susceptible to the parasites. Species in the genus Nosema O\r1icrosporidia: Nosematidae) are the most common entomopathogens with more than 150 described Nosema species found in 12 insect orders Olecnel and Andreadis, 1999).

"To whom correspondence should be addreslled. Email: gilsangjlgkhu.ae.k:r.

Since microsporidia lack mitochondria, they were considered to be extremely ancient eukaryotes (Vossbrinck et al., 1987). Evidence from protein coding genes, especially a- and jhubulins and phylogenetic analysis of microsporidia based on large subunit QStn ribosomal RNA (rRNA> sequences suggest that microsporidia share a common origin with fungi
analysis of microsporidia genome structure, Lee et al. (2()()B) consider the group a member of the kingdom Fungi. The sequences of microsporidian rRNAs are prokaryote-like and shorter than the known sequences

GENES & GENOMICS (2009) 31 : 443-450

444

ITS (37 bpI

5'

1=>-"'-'- - -S-S-U-(1-,2-36-b-~=>="'='- ~ ~ ¢='

¢='

1537R

HG4F!

-

LSU4F

LSU (2,506 bpI

I 3'

¢=' . . .

lLSUR LSU7R

Agure 1, Schematic diagram o! Nosema sp, COl rRNA gene. Mature rRNA gene domains are boxed. SSU rRNA gene was amplified by the primer set 18f/1537r (1236 bp); main parI 0' LSU rRNA gene was amplified by the primer set LS228F!ILSUR (2471 bp); the HG4F and HG4R primers were used to ampllfy Ihe partial SSU rRNA, ITS region and partial LSU rRNA genes (386 bpl. The arrows represent the 3' -end at each primer.

of eukaryotic or prokaryotic rRNA (Galtier and Gouy, 1995; Vossbrinck et aI" 1987). No distinct 5.88 rRNA gene has been found CYossbrinck and Woese, 1986; Gatehouse and Malone, 1998; Tsai et al., 2()()2). 'The small subunit (SSlJ) rRNA sequences of Microsporidia have been used to investigate the taxonomic position and phylogeny of microsJX)ridia (Vossbrinck et aI., 1987, 1993; Baker et aI., 1994; Baker et al., 1995; :Malone and McIvor 1996; Gatehouse and Malone. 1998; Wang et al., 2005; Kyei-Poku, et al., 2(08). In order to better understand the structural evolution of rRNA and taxonomy of Nosema species, the rRNA gene of four Nosema sp. isolates (COl, CO2,

C03 and C04) isolated from white cabbage butterfly,

Pieris rapae (Lepidoptera: Pieridae) in South Korea was investigated. Potential 2-dimentional structures of the SSU and LSU rRNA sequences of the isolate COl were analyzed. The results show that rRNA secondary structw-es may be used to explain relatedness among microsporidian species as suggested by Tsai et al. (2005). From the other three isolates, only SSU rRNA sequences were obtained. Phylogenetic analysis suggests that Nosema sp. COl, C02, C03, and C04 belong to the 'true' Nosema group. The members of the group are highly divergent within the Nosema! Vairimorpha clade

Table 1. Primers used for amplification and sequencing o! Nosema sp. rRNA.

Primer Small subunit rRNA (SSU) lSF 1537R

Internal transcribed spacer OTS) HG4F HG4R Large subunit rRNA (LSU) LS228F lUlUR Internal sequencing primers for LSU lBUF2 lBUF3 lBUF4 lBUR2 lBUR3 lBUR4 Specific primer for 3' end of LSU LSU7R

Sequence

Amplioon (bp) 1236

5'-CAC CAOOTI'GATICrGCC'3' 5'·TIA TGA TeC TCC TAA TCG TI'(}3' 386

5"GCG Gcr TAA TIT GAC TCA AC-3' 5"TCr ccr TOG TeC GTC 'IT1' CAk3' 2471

5'-GGA OOA AAA GAA ACT AAC-3' 5'-ACC TCT ere ACG ACG GTC TAA AC-3' 5'-CI'G ACG mc AAA TCG ATG A-3'

5'-CCG ATC TI'G GAG GCA GTA AC-3' 5'-MG ACC CTG TI'G AGC TI'G AC-3' 5'-TGA CTG CTG TCC TGA GAG AC-3' 5'·CI'G ATG COG TAT AGO TAC 00-3' 5"CCC AGC CM ACI' CCC ACT AT'3'

5'-TGG GTI TAG ACC GTC GTC AG-3' Primers 18F and 1537R are trom Vossbrinck el al. (1987). primers LS228F and ILSUR trom Vossbrinck ef al. (1993) and HG4F and HG4R from Galehouse and Malone (1998).

rRNA sequence and phylogeny of

Nosema isolates

MATERIALS AND METHODS Spore purification and genomic DNA preparation Spores of Nosema sp. COl were isolated from P.rapae (Choi et aI., 2002). The spores were purified using a gradient of neutralized Percoll (Sigma, USA) (Saoo and Watanabe, 1980). The purified spores were incubated in the digestion buffer with 400 ~ of proteinase K (Qj.agen, Germany) and 0.4 U of chitinase (Sigma, USA) at 56"C for 2 hrs followed by addition of gi= beads (500 ",,; diarne"', 425 W 600 """ Sigma, USA)
peR amplification, cloning and sequencing of the

rRNA gene

The primers used for rRNA gene amplification and sequencing are shown in Table 1. The amplification and sequence strategy is illustrated
445

with Pst I and Hind [I, and ligated to the Pst I I Hind III-digested DNA of pBluescript II SK-vector (Stratagene, Carlsbad, USA). The ligated DNA was used as template DNA The forward primer, ILSUF, was designed from the sequence of the concordant region of the reverse primer IlBUR (Table 1), and TI (5"TAA TAC GAC TeA eTA TAG GGC'3') was used as the reverse primer (Stratagene, USA). The genomic DNA (50 ng) was mixed in a 20 J.& reaction mixture containing PCR buffer [10 mM Tris'HCl, pH 9 0, 40 mM KCl, 15 mM MgCI,1, 250.,M of each dNTP, 20 pmole of each primer (Table 1), and 2.5 U of Taq DNA polymerase (Promega, USA). The PeR amplification was performed in a thermocycler (Biometra, Germany) for 35 cycles, each with the following profile; 95"C for 30 sec, 57'C for 30 sec, and 72'C for 1 min. After PeR amplification, the specific amplicon was obtained and sequenced on an automated DNA sequencer (AS! 377 Genetic Analyzer, Applied Biosystems, USA)

Secondary structure construction The secondary structures of Nosema sp. COl rRNA were constructed by using the RNA Viz program
446

GENES & GENOMICS (2009) 31 : 443450

phylogenetic tree construction was attempted with PAUP (ver 4.lOb, Swofford, 2(02) on 1000 bootstrap replicates. Tree-bisection-reconnection was employed as swapping algoritlun and branches less than 50'% of bootstrap support was collapsed. The consensus tree was visualized on 1'reegraph 2 (Millier and Miiller, 2(04).

.".. ....................\(... )~~ .....: ...:.,~ ..... .:'....,.~

.........

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'

SSU rRNA gene sequence The sequences of the SSU rRNA gene of Nosema spp. COl, CO2, C03 and C04 have been submitted to GenBank (accession number: AY383655, AY311589, AY311590 and AY311591 respectively). General information on the four SSU rRNA sequences can be found in Table 2. The secondary structure of the SSU rRNA of Nosema sp. COl shows that there is a core (formed by 1, 2, and 31 helices) and 4 branches (formed by 1-21, 22-30, 32-48, and 49-50 helices) from the 5' end clockwise to the 3' end. The helices 10, 18, and 46 are missing, and a helix localized between 23 and 24 helices specific to the eukaryotic model is numbered E23'1 (57 bp).

ITS sequence The ITS region of the Nosema sp. COl isolate, localized between SSU and LSD rRNA, contains only 37 bp (Genbank ace. No. AY383655). The base composition of the sequence is 8.1% G + C (data not shown). Even though the region from the transposed rRNA is similar to each other. the region is highly variable in its size from 26 to 185 bp and nucleotide composition when considering the region from outside of the 'true' Nosema group (data not shown). Therefore the region is not informative in constructing phylogenetic comparison as JXlinted. out by Thai et al. Table 2. Length and G+C (%) variation and divergence estimates among the tour SSU sequences of the isolates in this study.

Length G+C (bp)

(Q/\

,oJ

COl CO2 C03

1236 1233

34.22 34.31

H20

34.2

C04

1232

33.86

No. of base substitutions/sites COl

0.004 0.009 0.005

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C03

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2. Secondary structure model lor the SSU rRNA 'rom

Nosema sP. COl. Helices were numbered according to De Rijk et al. (2003). Red: 5' domain; green: central domain: orange: 3' malor domain; cyan: 3' minor domain.

(2005).

LSU rRNA gene sequence 100 l.SU rRNA gene of the Nosema sp. 0)1 oontains 2506 bp (Genbank ace. No AY3836SS) and the base composition of the lSU rRNA sequence is 33.tJillo G + C (data not shown). The secondary structure of the LSU rRNA gene of Nosema sp. COl shows that there are 7 groups (B to D distinguished clockwise from a core area and 12 hyper-variable areas (Vl-l2). Four helices (B7, 88, Bl4, and D5) are missing. Four areas of the hyper-variable regions are almost entirely missing (e.g. V2, VB, VlO and V12) and 4 areas are extremely reduced (e.g. Vl, V3, V5 and V6), based on the secondary structure of the eukaryotic lSU rRNA (De Rij k et al., 199Ba) (Fog. 3).

Phylogeny of SSU rRNA sequences of N0t18l1J8 sp. 0.014 0.006

0.014

COl, C02, C03, and C04 The final length of SSU rRNA used in phylogenetic

rRNA sequence and phylogeny of

Nosema isolates

Figure 3, secondary structure model lor lSU rRNA from Nosema sp, COl, Helix numbering is according fo De Rijk et al. (1998a). The boxed regions indicate parts of the structure where hypervariable areas are found in the eukaryotic rRNAs (De Rijk et al., 1998a; Wuyts el al., 2001).

447

448

GENES & GENOMICS (2009) 31 : 443-450

nD ,-EU219065 N. dis:;ln~' EU864526 N. antheraea8 05.e ,-AY]11591 Nosema sp. C04 '-AYJ11589 Nosema lip. C02 00996238 N empoascae U09282 H. bchop1lJ~i8e L39111 N. bombycis AY7473fJ7 N. spodoplerae' AY958071 N. pyrausta EU267796 V. ceraces EU219083 N. fumiferanae" AY960986 Nosema sp. PX\' AYJ83655 Nosema lip. C01. AYJ11S9Q Nosema sp. C03 AJ 131645 V. imperfecta EU 105211 Mtcrospondium sp. EU219082 Nosema sp. CPP'

EU338534 Nosema sp.· AY960987 N. pJutellae' AFl24331 Vauimorpha sp. 001. rV00266 V neea/fln

I--_'::':'+LU11051 V necarnx+

l

00996241 V neeattlK

l=J~~~~~X7389J

AJO lt833 N. granulO$Js

U2S532 N fumar;a.'is N aplS

AF327408 V. cheracis

EF585399 NOMlma sp. l39114 Vainmorpna sp.

.NosemaNa,nmorpna clade

• True

Nosema group

Figure 4. Maximum likelihood phylogenetic consensus tree (log !ikelihoocj; -3993.7222 al TN ratio of 1,3530, at Gamma distribution shape ot 0.5398) inferred from 30 partial SSU rRNA sequences 01 NosemalVairimorp/la clade with Vairimorpha sp. (Genbank acc. No. L39114) as an outgroup.

Genbank accession numbers are followed by scientific name and the isolates from this study are in a bold stroke. N. and It: represenl Nosema and Vairimorpha respectively. • represents LSU-ITS-SSU reverse arrangement and + SSU-ITS-LSU normal arrangement. Arrangement of rRNA of others is not known.

tree construction was 1378 bp. Based on the topology, the sequences of the four isolates in this study form a clade with other Nosema spp. that are previously classified in the 'true' Nosema group (Kyei-Poku et aI., 2008). Interestingly the Nosema sp. isolate COl is placed in the 'true' Nosema group, even though its rRNA arrangement is not reversed (Fig. 4). In addition species with other genus names such as Vairimorpah sp. V. ceraces, V. imperfecta, EndoreticuJatus sp. are placed in the 'true' Nosema group. Wang et aI.

(2005) reported that the EndoreticuJatus species belonged to the 'true' Nosema group and its SSU rRNA sequence identity was only 6()O/o of other EndoreticuJatus species. Therefore its nomenclature should be reconsidered. Likewise the Vairimorpha species in the 'true' Nosema group need to be reexamined (Kyei-Poku et aI., 2OOS).

DISCUSSION RNA molecules form a structure of helical regions interspersed with single stranded areas, the secondary structure of RNA molecules is very informative to fine-tune the alignment of sequences for phylogenetic studies
rRNA sequence and phylogeny of Nosema isolates microsporidian species may be more abundant than previously thought, as suggested in the phylogenetic tree. More sequence data as well as comparative analysis of NosemalVairimorpha clade rRNA SSU and LSU sequences and their secondary structure are required for high resolution insight into their taxonomic status.

ACKNOWLEDGEMENTS This work was supported by the Rural Development Administration fund, Republic of Korea. The authors would like to thank two anonymous reviewers for valuable conunents on the manuscripts.

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