Open Access

Analysis of a new strain of Euphorbia mosaic virus with distinct replication specificity unveils a lineage of begomoviruses with short Rep sequences in the DNA-B intergenic region

  • Josefat Gregorio-Jorge1,
  • Artemiza Bernal-Alcocer2,
  • Bernardo Bañuelos-Hernández1,
  • Ángel G Alpuche-Solís1,
  • Cecilia Hernández-Zepeda3,
  • Oscar Moreno-Valenzuela3,
  • Gustavo Frías-Treviño2 and
  • Gerardo R Argüello-Astorga1Email author
Contributed equally
Virology Journal20107:275

https://doi.org/10.1186/1743-422X-7-275

Received: 18 August 2010

Accepted: 19 October 2010

Published: 19 October 2010

Abstract

Background

Euphorbia mosaic virus (EuMV) is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep) and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country.

Results

A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR) led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade.

Conclusions

EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in the non-coding region of their DNA-B component, short rep gene sequences located downstream to a CP-promoter-like domain. This assemblage of DNA-A-related sequences within the DNA-B IR is reminiscent of polyomavirus microRNAs and could be involved in the posttranscriptional regulation of the cognate viral rep gene, an intriguing possibility that should be experimentally explored

Background

The members of the family Geminiviridae, one of the two largest natural groups of plant viruses, are characterized by a circular, single-stranded DNA (ssDNA) genome encapsidated within virions whose morphology is unique in the known virosphere, consisting of two joined, incomplete T = 1 icosahedra [1, 2]. Geminiviruses are classified into four genera, based on their genome organization, plant host range, and insect vector. Members of the most diversified genus, Begomovirus, are transmitted by the whitefly Bemisia tabaci (Hemiptera; Aleyrodidae), infect a wide range of dicotyledonous plant species, and have either monopartite or bipartite genomes [3]. In recent decades, these viruses have emerged as major threats to food and fiber crop production throughout the world, apparently as a result of a great increase in vector population densities, expansion of crop monocultures, transport of plant materials between geographically distant regions, and introduction of foreigner whitefly biotypes [4, 5].

Approximately 200 species of begomoviruses are currently known, grouped into two major lineages based on their genomic sequences: the Old World (OW; Europe, Africa, the Indian subcontinent, Asia, and Australasia) and the New World (NW; the Americas) begomoviruses [6, 7]. The OW begomoviruses have either monopartite or bipartite genomes, while all NW begomoviruses (for simplicity, NW-Beg) have two genomic components, known as DNA-A and DNA-B. The DNA-A component of NW-Beg has one open reading frame in the virion sense (AV1 or cp gene) encoding the coat protein, and four overlapped ORFs in the complementary sense (AC1 or rep gene, AC2 or trap gene, AC3 or ren gene, and AC4) that encode proteins involved in DNA replication, regulation of viral gene expression and suppression of host-defense responses [1, 8]. The DNA-B component contains only two ORFs, one in the virion sense (BV1 or nsp gene) and other in the complementary sense (BC1 or mp gene), encoding proteins involved in intra- and intercellular movement of the virus [9, 10]. The two genomic components are very different in overall nucleotide sequence, with the exception of a ~180-nt segment of the intergenic region (IR) displaying high sequence identity, termed the "common region" (CR). This region includes several repeated sequences (5 to 8-nt in length) called "iterons", which are closely associated to a ~30-nt conserved element that has the potential to form a hairpin structure that harbors in its apex the invariant nonanucleotide 5'-TAATATTAC- 3' [1]. Both the iterons and the conserved nonanucleotide in the hairpin element are functional targets for Rep, the virus-encoded protein that initiates the DNA replication by a rolling-circle (RCR) mechanism. Rep recognizes and binds specifically to the iterons and subsequently introduces a nick into the invariant nonanucleotide to initiate the RCR process [11, 12].

The NW-Beg have radiated to a great extent since its arrival to the American continent, and several secondary lineages or "clades" have been identified in phylogenetic studies [6, 13, 14]. The most atypical of the NW-Beg clades is the one named after the Squash leaf curl virus (SLCV) that encompasses more than 15 viral species distributed from Southern EUA to Brazil [7, 13]. Members of the SLCV clade are differentiated from other NW-Beg by two main features: 1) the number and arrangement of the iterons in their replication origin, that are distinctive, and 2) the N-terminal domain (i.e., residues 1 to 150) of their Rep proteins display low aa sequence identity (< 50%) with proteins encoded by typical NW-Beg, lacking several amino acid motifs which are conserved in both NW- and OW- begomovirus Rep proteins [[1517]; unpublished data].

Among the earliest recorded members of the SLCV-clade is Euphorbia mosaic virus (EuMV), which was associated with symptomatic Euphorbia heterophylla plants throughout the Caribbean basin and the tropical Americas since the 1970's [18, 19]. However, its molecular characterization was not carried out until 2007, when the complete genome sequence of EuMV-YP, the isolate associated with the former plant host in the Yucatan Peninsula of Mexico, was reported [20]. Complete DNA-A sequences from two additional EuMV isolates were available at GenBank at that time, one from Puerto Rico (EuMV-PR) and the isolate whose complete sequence is now reported here, from Jalisco, Mexico (EuMV-Jal). According to their full-length DNA-A sequence identity, the EuMV isolates were classified into two different strains, simply termed "A" and "B". The first strain was represented by EuMV-YP and EuMV-PR, while EuMV-Jal was the only member of the "B-strain" [7]. However, the recently described EuMV-JM, from Jamaica [21], displays a very similar sequence identity to both EuMV-PR (A-strain, 95% identity) and EuMV-Jal (B-strain, 95.4% identity). Therefore, the relationship between EuMV isolates belonging to supposedly distinct strains should be experimentally addressed.

In this work we report the complete molecular characterization of EuMV-Jal, which was found infecting peppers and weeds in Jalisco, Mexico, and was shown to be incompatible in replication with EuMV-YP in reassortment experiments. The genomic analysis of this novel EuMV strain led to the unforeseen discovery of an assemblage of DNA-A homologous sequences in the intergenic region of its DNA-B, whose position and arrangement is conserved in several begomovirus species, hence suggesting the intriguing possibility of a functional role of those atypical sequences in the infective cycle of EuMV and its relatives.

Results

Isolation of a new strain of Euphorbia mosaic virus

During Autumn 2005, a survey of farming fields infested with whiteflies in the state of Jalisco, Mexico, was undertaken. Pepper plants exhibiting a variety of symptoms (including leaf curling and crumpling, yellow veins, deformed fruits, and stunted growth) were observed in fields of three Jalisco localities. Leaf samples from 63 symptomatic weeds and pepper plants were collected, and total DNA extracts were tested for the presence of begomoviruses using polymerase chain reaction (PCR) with several pairs of degenerated primers (see Methods). More than 80% of the examined samples were PCR-positive and sequence analyses of the amplicons revealed that the majority of the symptomatic plants were infected by begomoviruses belonging to two different species, Pepper huasteco yellow vein virus (PHYVV) and Pepper golden mosaic virus (PepGMV), which commonly infect pepper and tomato crops throughout the north and central areas of Mexico [2224]. Partial DNA-A sequences of a third begomovirus were obtained from two pepper samples from the Castillo locality (close to the Pacific coast, coordinates 19°45'00'' N; 104°23'30'' W), one Nicotiana glauca plant ("tabaquillo") collected at Sayula (coordinates 19° 47'55'' N; 103°46'05'' W) and one Euphorbia heterophylla plant collected at Teocuitatlán (coordinates 20°12'30'' N; 103°30'00'' W). In the four cases the plants were co-infected with either PHYVV or PepGMV. The complete sequence of the DNA-A and DNA-B genomic components of the unidentified begomovirus was obtained from overlapped PCR products derived from one pepper plant co-infected with PHYVV (see Methods). Comparisons with sequences available at the GenBank database using BlastN showed that the third pepper-infecting virus was an isolate of Euphorbia mosaic virus, displaying a DNA-A overall sequence identity of 95.4%, 92.8% and 92.1% with EuMV isolates from Jamaica [GenBank: DQ395342], Puerto Rico [GenBank: AF068642] and the Yucatan Peninsula [GenBank: DQ318937], respectively.

Genome organization of EuMV-Jal

The EuMV-Jal genome exhibited a genetic organization typical of NW-Beg. The DNA-A molecule [GenBank: DQ520942] was 2609 nt in length, and encoded five genes (cp, rep, trap, ren and AC4). The DNA-B molecule [GenBank: HQ185235] was 2590 nt in size, and contained two major ORFs (BV1 and BC1). The common region (CR) of EuMV-Jal DNA-A and DNA-B encompassed 169 and 170 nt, respectively, with 98% identity. The CR contained the origin of replication comprising the conserved hairpin element and five iterons (GGAGTCC) that displayed the characteristic arrangement of the viruses belonging to the SLCV-cluster [15, 16]. Comparisons of EuMV-Jal CR with the homologous region of other EuMV isolates revealed that EuMV-Jal and EuMV-JM have a DNA-A replication origin with a composition of putative cis-acting elements different to the homologous Ori of EuMV-YP and EuMV-PR. Indeed, in addition to harbor iterative elements with a distinct nucleotide sequence, the EuMV isolates from Jalisco and Jamaica display a G-box motif in the immediate vicinity of the conserved hairpin element, which is absent in the DNA-A of EuMV-PR and EuMV-YP (Figure 1A). The later viruses display instead a conserved motif (GGGGCAAAA) that is characteristic of most members of the SLCV-clade (our unpublished data). In contrast with the differences observed between the DNA-A components, comparisons of the DNA-B CR revealed a similar modular organization in all EuMV isolates, with a G-box motif adjacent to the hairpin element (Figure 1B). A similar organization of the DNA-B CR is observed in Euphorbia yellow mosaic virus (Fernandes et al., unpublished) [GenBank: FJ619507 and FJ619508], a recently described begomovirus from Brazil, that is a distant relative of EuMV (Figure 1B).
Figure 1

Comparison of CR sequences from EuMV and relatives. The alignments of the CR sequences of both (A) DNA-A and (B) DNA-B components from EuMV isolates and related begomoviruses from South America are shown to highlight similarities and differences in relevant cis-acting elements. Putative Rep-binding elements (iterons) are shaded in yellow and their relative orientation is depicted by black arrows; the sequence with the potential to form a stem-loop structure is highlighted in black and underlined. The TATA box of the leftward promoter is shaded in blue. The G-box element is shown in red letters, and the "GYA box" conserved in members of the SLCV clade is represented in green letters. (C) Differences in the nucleotide sequence of the iterons and the amino acid sequence of the Rep-IRD of EuMV-Jal and relatives are highlighted. Virus acronyms and GenBank accession numbers are listed in Table 1.

Phylogenetic relationships

A phylogenetic tree based on the full-length DNA-A of four EuMV isolates, 20 NW-Beg and several bipartite and monopartite OW-Beg (Table 1), was generated using the neighbor-joining method with 1,000 bootstraps replications (Figure 2). The analysis indicated a close relationship between the EuMV isolates from Mexico and the Caribbean basin with the following three begomoviruses from South America: Tomato mild yellow leaf curl Aragua virus (TMYLCAV) from Venezuela [GenBank: AY927277], Euphorbia mosaic Peru virus (EuMPV) [25], and Euphorbia yellow mosaic virus (EuYMV) from Brazil. This grouping was well-supported by both the phylogenetic analysis (bootstrap value 84) and the pairwise-identity analyses (Table 2), thus defining a sub-lineage within the SLCV clade that is broadly distributed in the American continent. A phylogenetic analysis based on the full-length DNA-B sequences produced similar results for the EuMV subclade and the group of cucurbit-infecting viruses (data not shown), but not for other members of the SLCV lineage that were placed into groups that are not congruent with the phylogeny derived from their DNA-A sequences. The incongruent phylogenies of DNA-A and DNA-B components of some begomoviruses is generally indicative of recombination and/or reassortment events [6, 26].
Table 1

Names, acronyms, and GenBank accession numbers of the geminiviruses used in this study

Name

Acronym

Accession number

  

DNA-A

DNA-B

Abutilon mosaic virus

AbMV

NC_001928

NC_001929

African cassava mosaic virus

ACMV

NC_001467

NC_001468

Ageratum yellow vein virus

AYVV

NC_004626

 

Bean calico mosaic virus

BCaMV

NC_003504

NC_003505

Bean dwarf mosaic virus

BDMV

NC_001931

NC_001930

Bean golden yellow mosaic virus

BGYMV

NC_001439

NC_001438

Beet curly top virus

BCTV

NC_001412

 

Beet mild curly top virus

BMCTV

NC_004753

 

Cabbage leaf curl virus

CabLCV

NC_003866

NC_003887

Chino del tomate virus

CdTV

NC_003830

NC_003831

Corchorus golden mosaic virus

CoGMV

NC_009644

NC_009646

Corchorus yellow vein virus

CoYVV

NC_006358

NC_006359

Cotton leaf crumple virus

CLCrV

NC_004580

NC_00481

Cotton leaf curl multan virus

CLCuMV

NC_004607

 

Cucurbit leaf crumple virus

CuLCrV

NC_002984

NC_002985

Desmodium leaf distortion virus

DeLDV

NC_008494

NC_008495

Euphorbia leaf curl virus

EuLCV

NC_005319

 

Euphorbia leaf curl India virus

EuLCIV

EU194914

 

Euphorbia mosaic Peru virus

EuMPV

AM886131

 

Euphorbia mosaic virus-Jalisco

EuMV-Jal

DQ520942

HQ185235

Euphorbia mosaic virus-Jamaica

EuMV-JM

FJ407052

EU740969

Euphorbia mosaic virus-Puerto Rico

EuMV-PR

AF068642

 

Euphorbia mosaic virus- Yucatan

EuMV-YP

NC_008304

NC_008305

Euphorbia yellow mosaic virus

EuYMV

NC_012553

NC_012554

Papaya leaf curl virus

PaLCuV

AJ436992

 

Pepper golden mosaic virus

PepGMV

NC_004101

NC_004096

Pepper huasteco yellow vein virus

PHYVV

NC_001359

NC_001369

Rhynchosia golden mosaic Yucatan virus

RhGMYucV

NC_012481

NC_012482

Sida golden mosaic virus

SiGMV

NC_002046

NC_002047

Squash leaf curl virus

SLCV

NC_001936

NC_001937

Squash mild leaf curl virus

SMLCV

NC_004645

NC_004646

Squash yellow mild mottle virus

SYMMoV

NC_003865

NC_003860

Tomato common mosaic virus-Brazil

ToCoMV-BZ

NC_010835

NC_010836

Tomato golden mosaic virus

TGMV

NC_001507

NC_001508

Tomato mild yellow leaf curl Aragua virus

TMYLCAV

NC_009490

NC_009491

Tomato mottle virus

ToMoV

NC_001938

NC_001939

Tomato severe leaf curl virus

ToSLCV

DQ347947

 

Tomato yellow leaf curl Thailand virus

TYLCTHV

X63015

X63016

Tomato yellow leaf curl virus

TYLCV

X15656

 

Watermelon chlorotic stunt virus

WmCSV

NC_003708

NC_003709

Figure 2

Phylogenetic relationships of Euphorbia mosaic virus. The tree was constructed using Neighbor-joining algorithm implemented by MEGA4 software (66). Branch strengths were evaluated by constructing 1000 trees in bootstrap analysis by step-wise addition at random. Bootstrap values are shown above or under the horizontal line. The vertical distances are arbitrary, whereas the horizontal distances are drawn to scale with the bar indicating 0.05 nucleotide replacements per site. Curtoviruses (Beet curly top virus and Beet mild curly top virus) were used as out-groups. Virus acronyms and GenBank accession numbers are listed in Table 1.

Table 2

Percentages of sequence identities between EuMV-Jal and selected begomoviruses (DNA and predicted proteins*)

 

DNA-A

IR-A

CP*

AC1*

AC2*

AC3*

AC4*

DNA-B

IR-B

BV1*

BC1*

Virus

           

ACMV

45

25

66

49

43

42

19

27

22

24

41

BCaMV

76

50

92

86

78

77

64

55

28

73

83

BGYMV

64

37

91

63

70

78

11

48

22

67

80

CdTV

67

43

92

63

67

78

30

51

27

71

78

CoYVV

51

24

87

43

51

43

19

41

22

52

71

CuLCrV

77

46

91

83

71

71

72

51

27

66

76

DesLDV

72

44

91

80

66

73

58

50

23

64

77

EuMPV

77

52

93

86

81

76

58

-

-

-

-

EuYMV

77

51

90

85

80

76

62

52

35

73

82

EuMV-JM

95

91

98

97

97

95

88

86

73

96

98

EuMV-PR

92

82

99

96

93

91

91

-

-

-

-

EuMV-YP

92

80

99

93

93

91

87

85

63

94

98

PepGMV

72

50

90

80

71

75

14

48

25

64

74

PHYVV

59

33

89

49

50

63

12

47

25

66

74

RhGMYV

76

54

94

86

70

70

66

51

31

69

78

SLCV

78

57

94

82

72

80

77

50

30

63

80

ToCoMV-BZ

73

43

90

85

64

72

57

52

31

63

77

TMYLCAV

84

66

95

88

87

80

82

56

43

75

83

TYLCTHV

48

28

68

48

43

39

22

25

19

21

39

Recombination analysis

The differences between the strains A and B of EuMV regarding nucleotide sequence and modular organization of the Ori region could be indicative of either divergent molecular evolution or intermolecular recombination between co-infecting begomoviruses [27, 28]. To search for potential recombinant sequences in the genome of EuMV strains, we analyzed sequence alignments that included the DNA-A of the four EuMV isolates under exam, as well as diverse sets of begomoviruses of the SLCV clade, using the suite of programs for detection of recombinant breakpoints integrated within the RDP package [29]. The analysis identified a ~210-nt long EuMV genomic region (recombinant breakpoints at positions 2432 and 33 of EuMV-Jal DNA-A) as a fragment of possible recombinant origin, which includes the entire common region (~ 170-nt) as well as the first 44 nucleotides of the rep gene, encompassing the IRD-coding sequence [17]. The plausible recombinant origin of this DNA fragment is underscore by direct comparisons of the DNA-A components from EuMV-JM and EuMV-PR, which are members from different strains exhibiting very high sequence identity (97.4%) along a segment encompassing ~2,400 out the 2,609-nt of its DNA-A, a fact that is in clear contrast with the low sequence identity (77.5%) displayed in the 210-nt genomic region flanked by the recombinant breakpoints detected by our analysis.

The assembled data suggest that EuMV A-strain viruses are the product of an intermolecular recombination event involving an EuMV-JM-related virus (the major parent) and a virus closely related to Calopogonium golden mosaic virus (CpGMV) [GenBank: AF439402] which might have donated the ~210-nt fragment with the viral replication module. This DNA segment, which is entirely identical in sequence between EuMV-PR and EuMV-YP, is shared with CpGMV at 90% of nucleotide identity. Two additional observations support the hypothesis of intermolecular recombination: (1) The absence of a G-box element within the CR of the DNA-A component of EuMV-YP, that is nevertheless present in their cognate DNA-B component (see Figure 1); and (2) The lower than expected sequence identity of the EuMV-YP common region (i.e., 86%) that is in contrast with the high identity of the CR of both EuMV-Jal and EuMV-JM (98% and 96%, respectively) [20, 21].

Experimental infection of host plants

EuMV-Jal was identified in four field samples that contained an additional, distinct begomovirus, as mentioned above. In order to examine experimentally EuMV-Jal in single plant infections, we generated infectious clones of both DNA-A and DNA-B components (see Methods), and carried out biolistic inoculation of these clones into four plant species: Datura stramonium, Nicotiana benthamiana, pepper (Capsicum annum), and zucchini (Cucurbita pepo). All solanaceous species were susceptible and developed systemic symptoms at 10-12 dpi, while the zucchini plants did not show symptoms and no viral DNA was detected by PCR in their tissues at 14 dpi. Symptoms of EuMV-Jal infection varied between plant species. In N. benthamiana the symptoms included leaf crumpling, greenish mosaics and shortened internodes (Figure 3A). In pepper plants the first symptom was the appearance of small green spots that progressed into a pale green mosaic and moderate downward leaf curling; a few small necrotic spots were also observed in several plants (Figure 3B). The most severe symptoms were observed in D. stramonium plants, whose leaves showed deformation and extensive green and yellow mottle covering most of the foliar surface, progressing in time to necrotic lesions leading to the destruction of significant parts of the foliar lamina (Figure 3C). In all, the symptoms induced by EuMV-Jal in the examined three plant species were very similar to those generated by infection of EuMV-YP [20], hence suggesting that these viruses express equivalent pathogenesis factors, as expected from the high amino acid sequence identity of their predicted proteins (Table 2).
Figure 3

Symptoms induced by EuMV-Jal in experimentally infected plants. (A) Nicotiana benthamiana, (B) Capsicum annum, and (C) Datura stramonium.

EuMV-Jal and EuMV-YP are incompatible in replication

The replication modules of the EuMV strains A and B exhibit two main differences: 1) their iterons display a different nucleotide N within the GGNGTCC core, and 2) the iteron-related domain of their Rep proteins have a different amino acid residue at position X3 of the IRD core FX1L*X3[17], that is either FRLA or FRLT in A-strain viruses, and FRLQ in B-strain members (Figure 1C). These observations suggest the intriguing possibility that EuMV strains A and B could be incompatible in replication. To answer this question we carried out reassortment experiments with the EuMV-Jal and EuMV-YP genomic components. The four possible combinations A+B of the cloned viral DNAs were biolistically inoculated into N. benthamiana plants, that were subsequently scored for the appearance of disease signs.

Systemic symptoms developed at 10-12 dpi in most plants inoculated with the homologous mixtures (i.e., EuMV-Jal [A+B], and EuMV-YP [A+B]); in contrast, the plants bombarded with the heterologous combinations (i.e., EuMV-Jal [A]/-YP [B] and its reciprocal, EuMV-YP [A]/-Jal [B]) displayed no symptoms at 12 dpi, and remained symptomless until the end of the experiment, at 30 dpi (Figure 4A). These experiments were repeated three times, six plants for each combination, with similar results obtained (data in Figure 4B). All plants inoculated with cognate viral components scored positive for presence of both EuMV DNA-A and DNA-B, based on PCR detection of a ~1300-bp fragment encompassing a part of the rep and cp genes and the entire DNA-A intergenic region, and a ~1400-bp segment comprising the DNA-B IR and a part of both BV1 and BC1 genes. In contrast, none of the newly emerged leaves of plants bombarded with the heterologous combinations of EuMV genomic components tested positive for presence of EuMV DNA-B, although a few plants (5 out 36) were PCR-positive for DNA-A at 14 dpi, but not at 28 dpi (data not shown). These results indicate that viral factors required for replication are not exchangeable between EuMV-Jal and EuMV-YP.
Figure 4

EuMV-Jal does not form viable reassortants with EuMV-YP. (A) N. benthamiana plants inoculated with either the two genomic components of EuMV-Jal (left), or the heterologous combination EuMV-Jal DNA-A/EuMV-YP DNA-B (right). Plants were inoculated by microparticle bombardment with 5 μg of each DNA component, and photographed 26 days after inoculation. (Panel B) Results of the reassortment experiments between EuMV-YP and EuMV-Jal. Negative controls (plants inoculated with the empty vector) were included in the three independent experiments but the data were omitted for simplicity.

EuMV BV1 promoter contains a short sequence homologous to Rep gene

In the course of a meticulous scrutiny of the DNA-B intergenic region of EuMV-Jal to identify potential cis-regulatory elements involved in the transcriptional control of the BC1 and BV1 genes, we unexpectedly discovered a 35-bp DNA stretch displaying 100% sequence identity with a segment of the homologous rep gene. This sequence is located ~150-nt upstream to the BV1 gene (nucleotides 337-372) and contains the coding information for aa residues 15 to 25 of EuMV-Jal Rep (i.e., FLTYPQCDVPK) that includes the conserved Motif I of the RCR initiators [30]. No additional sequences homologous to the rep gene were found in the BV1 promoter region. The finding of a short sequence apparently derived from the cognate DNA-A within the noncoding region of EuMV-Jal DNA-B was intriguing and prompted further scrutiny of other EuMV DNA-B components. In all the examined cases a short Rep homologous sequence (sRep HS) was found within the BV1 promoter region, which in EuMV-JM is similar to the EuMV-Jal element in both sequence and length (35-nt), but that is longer in EuMV-YP that displays a DNA stretch 51-nt in length identical to a segment of its cognate rep gene (Figure 5). A search for analogous elements in the DNA-B IR from all members of the SLCV clade revealed that sRep HS elements are not common, being identified only in two close relatives of EuMV, namely, TMYLCAV from Venezuela and EuYMV from Brazil. The TMYLCAV sRep HS element is similar but not identical in both length (36-nt) and nucleotide sequence (88% identity) to the equivalent sequence of EuMV-Jal (Figure 5). In contrast, the sRep HS identified in EuYMV DNA-B is different in both length (45-nt) and nucleotide sequence (< 30% identity) to the analogous elements of EuMV strains. Indeed, the EuYMV sRep HS element corresponds to a distinct segment of the cognate rep gene, encoding the Rep aa residues 40-53 (i.e., VVKPTYIRVARELH) instead of Rep residues 15-25 encoded by the sRep HS elements of TMYLCAV and EuMV. Notwithstanding its divergent nucleotide sequence, the EuYMV sRep HS element is 100% identical in nucleotide sequence to a segment of its cognate rep gene, like in EuMV and TMYLCAV (Figure 5) and is located at a position equivalent to the sRep HS in the latter viruses.
Figure 5

Nucleotide sequence of s Rep HS elements. The upper sequence correspond to the DNA-B and the lower one to the cognate DNA-A. Letters in red within the sRep HS elements of EuMV-YP and TMYLCAV denote differences with the homologous sequence of EuMV-Jal.- Virus acronyms are listed in Table 1.

sRep HS upstream sequences are similar to CP promoters

The conservation of sRep HS elements in the DNA-B intergenic region of EuMV and their relatives suggests that those atypical sequences might play a defined role in the infective cycle of these viruses. Since the sRep HS elements do not contain a start codon and are not a part of a distinctive ORF, it seems plausible that its function, if any, involves an intermediary RNA molecule. This notion naturally led us to suggest the existence of a functional promoter next to the sRep HS element.

In order to identify potential IR internal promoters, we analyzed the sequences upstream to sRep HS in all members of the EuMV lineage using a phylogenetic-structural approach. This methodology entails the identification of "phylogenetic footprintings" (i.e., putative binding sites for transcription factors) and conserved arrays of them, named "Conserved Modular Arrangements" (CMAs), in non-coding regions of evolutionarily-related DNA sequences [31, 32]. The new analysis exposed a DNA-B IR domain ~160-bp-long exhibiting a remarkable similarity both in overall nucleotide sequence and modular organization, to CP promoters of viruses that belong to the SLCV clade. The example showed in Figure 6 illustrates the remarkable similarity between the CP promoter-like (CP prom-L) domain of EuMV-Jal IR and a 156-nt segment of the CP promoter of Rhynchosia golden mosaic Yucatan virus (RhGMYuV), a recently described virus of the SLCV lineage [33]. The similarity between these DNA-B and DNA-A sequences, respectively, includes nine phylogenetic footprintings in a definite order, and it is extended beyond the start codon of RhGMYuV cp gene including a block of 8-nt of coding sequence that is conserved in the non-coding sequence of EuMV-Jal DNA-B.
Figure 6

Potential internal promoter in the DNA-B intergenic region of EuMV. Alignment of a domain of the DNA-B intergenic region of EuMV-Jal with the coat protein (CP) gene promoter of Rhynchosia golden mosaic Yucatan virus [GenBank: GQ352453]. The blocks of high sequence similarity encompassing more than eight nucleotides are "phylogenetic footprintings" (PFp; boxed). Putative cis-acting elements within the PFp (identified by roman numerals) are as follows: I) Conserved Late Element, CLE (inverted); II) salycilic acid responsive element (inverted, restricted similarity); III) A pathogen-elicitor responsive element (Box W1) overlapped with a CCAAT box; IV) conserved element with indeterminated function; V) sequence highly similar to the 3'end of the TGMV CP promoter "region C", that functions as transcriptional negative element [67]; VI) potential auxin-responsive element (ARE, at the block 3'end); VII) TATA box; VIII) canonical salycilic acid responsive element; IX) ethylene-responsive element (ERE)-like motif. The block "X" includes the first segment of the RhGMYuV CP gene. Notice the 8-nt long stretch of identical sequence between the non-coding sequence of EuMV-Jal DNA-B and the coding sequence of RhGMYuV.

The demarcated CP prom-L domain of the DNA-B IR includes several putative cis-regulatory elements that were identified by consulting plant transcription factors databases like PlantCare [34] and PLACE [35]. Among the identified potential cis-acting motifs there were well-characterized regulatory elements such as the "Conserved Late Element" (CLE) [36], the CCAAT box, and several elements that confer responsiveness to a variety of plant hormones (see Figure 6 legend). Among these sequences there is a 12-bp long element (consensus: CTTTAATTCAAA) which is identical to a conserved sequence immediately adjacent to the cp gene in more than 75% of the known begomoviruses from America (Cardenas-Conejo et al., unpublished data). The AATTCAAA motif of the former element is both a putative ethylene-responsive element (ERE) and a binding-site for nuclear factors of carnation, tomato and Solanum melongena[3739]. In addition, this motif constitutes the 8-nt long leader sequence of the CP mRNA of Tomato golden mosaic virus (TGMV) [40]. The ERE-like motif is located downstream to the actual TATA-box of NW-Beg CP promoters, at a similar distance (21-29 bp) to that observed between the ERE and a putative TATA box in the CP prom-L domain [Additional file 1: Supplemental Figure S1a]. Taken as a whole, these remarkable similarities between noncoding DNA regions from two different genome components of separate begomovirus species, can hardly be explained by random sequence convergence; rather, they strongly suggest that the DNA-B CP prom-L domain of EuMV and relatives is evolutionarily derived from a begomovirus CP promoter.

Distantly related begomoviruses contain sRep HS elements

The existence of sRep HS elements in the DNA-B IR of viruses belonging to a minor lineage of the SLCV clade is an interesting evolutionary enigma. To determine whether analogous elements actually exist in other viral lineages, we searched for rep homologous sequences in the DNA-B IR of begomoviruses belonging to 12 major and minor clades, distributed in several continents. The analysis of ~60 members of those lineages led us to the identification of only two additional begomoviruses displaying sRep HS in the BV1 upstream region: TGMV and the recently described Cleome leaf crumple virus (ClLCrV) [41]. These viruses are native from Brazil, like EuYMV, but do not belong to the SLCV clade. The sRep HS element of ClLCrV is 100% identical to a 46-nt-long segment of its cognate rep gene, encoding the aa residues 97 to 110 (SSSDVKSYVDKD GD), that comprise the conserved RCR Motif 3 (underlined) [30]. On the other hand, the TGMV sRep HS element is only 88% identical to a 52-nt-long segment of its cognate rep gene, encoding the aa residues 255 -271 (NKVEYNVIDDV TPQYLK) of this replication initiator, that include the Walker B-motif (underlined), a critical aa sequence of the protein ATPase/helicase domain [42, 43].

The upstream sequences of TGMV and ClLCrV sRep HS elements were examined, but no significant similarity between them nor with the BV1 promoter region of EuMV lineage viruses was found. However, a careful re-examination of sequences nearby to the 5'end of ClLCrV sRep HS revealed a 23-bp sequence with partial dyad symmetry that is well-conserved both in sequence and in position relative to the sRep HS element in all viruses of the EuMV cluster [Additional file 1: Suppl. Figure S1b]. The consensus of this conserved sequence includes a palindromic core with the repeated motif TTGTGGTCC, similar to the CLE, a functional target of plant transcriptional activators [44, 45] that has been involved in TrAP-mediated activation of the CP promoter in some begomoviruses [36]. No sequence similar to the latter symmetric element was found in the BV1 promoter region of TGMV. In fact, the sRep HS of the latter virus differs from the analogous elements in ClLCrV and the EuMV subclade viruses in several other important features: (1) It is not 100% identical to the corresponding segment of its cognate rep gene; (2) It has opposite polarity compared to all other known sRep HS elements; (3) It is closely located downstream to a putative internal promoter that does not exhibit significant similitude with CP promoters of SLCV clade viruses (data not shown). It is relevant to point out here that TGMV and ClCrV are grouped, on the basis of their full-length DNA-A sequences, within the Brazilian cluster of NW-Beg [41], but they have very divergent DNA-B components. Thus, our finding of the sRep HS-associated semi-palindromic sequence in ClLCrV DNA-B suggests an actual relationship of the latter with the homologous genomic components of EuMV and relatives, a notion that is supported by a recent study that groups the ClLCrV DNA-B with viruses of the EuMV lineage [41].

Discussion

In this study, we described the molecular and biological characterization of a novel strain of Euphorbia mosaic virus that was isolated from pepper plants in the state of Jalisco, Mexico, near to the Pacific shoreline. This virus displays 92% sequence identity with EuMV-YP, that was isolated in the same country but in a distant region, close to the Atlantic coastline [20]. These viruses differ in two important features of their DNA-A replication origin region: the nucleotide sequence of their iterons, and the presence or absence of a G-box element, a cis-acting sequence which is critical for Rep promoter activity in some NW-Beg [46]. The differences observed in the predicted Rep-binding sites of EuMV-Jal and EuMV-YP prompted us to explore experimentally their ability to form viable reassortants in pseudorecombination tests. The results of these experiments confirmed the presumption of replication incompatibility between EuMV-YP and EuMV-Jal, thus demonstrating that the latter is a new, biologically-defined strain exhibiting different replication specificity.

The finding of begomovirus strains that are not able to form viable reassortants is somehow bewildering because the common definition of a virus species is "A...class of viruses that constitutes a replicating lineage and occupies a particular ecological niche." [47, 48]. Accordingly, it is not expected that strains of a virus species would be incompatible in replication because that implies that they do not constitute an actual replicating lineage. Nonetheless, it is generally recognized that several strains of begomoviruses probably are not complementary in replication because they display different putative cis- and trans-acting replication specificity determinants [7, 17]. There is at least one report of strains belonging to a bipartite begomovirus that are not equivalent in replication functions (the "severe" and "mild" strains of Tomato leaf curl New Delhi virus, ToLCNDV) [49]. However, that case is different from the one examined here because the "mild" phenotype of one ToLCNDV strain seems to be related to an inefficient trans-replication of the "cognate" DNA-B, which displays Rep binding-sites different to those of the associated DNA-A [49, 50].

The case of the EuMV strains is significant because it is paradigmatic of an apparently common theme in begomovirus evolution, i.e., the sudden change of virus replication specificity determinants by intermolecular recombination between co-infecting viruses [27, 51]. Indeed, the recombination analysis of EuMV isolates indicates that viruses of the EuMV A-strain probably evolved by an event of DNA intermolecular exchange involving a member of the EuMV B-strain and a virus related to CpGMV, which had donated a ~210-bp DNA segment encompassing the region of the virus replication origin and the first 44 nucleotides of the rep gene. If this hypothetical scenario is accurate, then the recombination event should have changed simultaneously both the iterons and the Rep aa residues interacting with them, thus maintaining the proper matching of cis- and trans-acting replication determinants in the recombinant DNA-A component.

Diverse studies have identified the sequences encompassing the viral strand replication origin and the rep gene segment encoding the Rep N-terminal domain, as the regions of geminivirus genomes most frequently exchanged during recombination [28, 5153]. This is consistent with the known genome localization of the Rep-binding sites and the coding sequence of the Rep domain that contains the putative DNA-binding specificity determinants of this protein, which have been theoretically mapped into the first 75 aa residues [17, 54]. Consequently, a recombination event involving a genome portion as small as 200 to 360-bp might confers a completely different replication phenotype to begomoviruses involved in mixed infections, as presumably is the case for the EuMV strains.

Since that intermolecular recombination is/has been a major force in the evolution of geminiviruses, the concepts of both "species" and "strains" should be adapted to the peculiar nature of these entities, that are genetic mosaics in continual change, different in quality to cellular organisms. In fact, it is altogether possible that a significant part of the currently recognized begomovirus species would not constitute "replicating lineages" in a strict sense, as would be the case of EuMV, according to our experimental data. For instance, a thorough sequence analysis entailing the identification of the putative cis- and trans-acting Replication Specificity Determinants (RSDs) of the 182 recognized begomovirus species summarized by Fauquet et al. in 2008 [7] revealed the existence of 34 species that include at least two groups of viruses exhibiting distinct putative RSDs, analogous to the strains A and B of EuMV. Furthermore, some ICTV-accepted species as Ageratum yellow vein Hualian virus, Honeysuckle yellow vein virus, Tomato leaf curl Bangalore virus, Tomato leaf curl Philippines virus, Tomato leaf curl Taiwan virus, and ToLCNDV, include three classes of viruses differing in their putative RSDs, and one viral species, Ageratum yellow vein virus, comprises four types of viruses harboring distinct replication modules, plausibly acquired through independent episodes of intermolecular recombination (Arguello-Astorga, unpublished data). In view of the significant number of begomovirus species with variants that are seemingly analogous to the strains of EuMV, it would be important to establish a formal distinction between strains with similar RSDs, that represent actual replicating lineages, and replication-incompatible strains, that apparently do not.

What is the function of the DNA-B sRep HS elements?

During the analysis of the intergenic region of EuMV-Jal DNA-B we discovered a short DNA stretch identical to a segment of the rep gene coded in the cognate DNA-A. It was subsequently find out that analogous sRep HS elements exist in the DNA-B IR of at least five begomovirus species, all them from the New World: EuMV from Mexico and the Caribbean basin, TMYLCAV from Venezuela, and EuYMV, ClLCrV and TGMV from Brazil. With the exception of the short rep homologous sequence in the DNA-B IR of TGMV (that seems to be evolutionarily unrelated) the sRep HS elements of begomoviruses have in common several characteristics. All of them: (1) are short sequences, ranging from 35 to 51 nucleotides in length; (2) are 100% identical in nucleotide sequence to a segment of its cognate rep gene; (3) have opposite polarity than the rep gene; (4) are located 65 to 80-nt downstream to a putative internal promoter highly similar to CP promoters of viruses of the SLCV clade (ClLCrV being an exception); (5) are positioned 7-9 nt downstream to a 23-bp partly palindromic element with a repeated motif similar to the CLE; and (6) are situated 115 to 145-nt upstream to the BV1 gene. In contrast, the sRep HS elements of viruses that are distantly related, like EuMV, EuYMV and ClLCrV, have entirely different nucleotide sequences (see Figure 5), because the coding sequence represented in those elements corresponds to distinct sections of the cognate rep gene.

An intriguing observation is that the identified sRep HS elements reproduce sequences encoding conserved aa motifs which are critical for Rep functions. For example, the sRep HS of EuMV strains and TMYLCAV correspond to the coding sequence of RCR Motif 1; the equivalent element of ClLCrV encodes the RCR Motif 3, and the analogous sRep HS of TGMV duplicate the rep sequence encoding the Walker B motif of ATPases/helicases. An apparent exception is the sRep HS of EuYMV, which displays the coding sequence of a conserved Rep motif of unknown function. The evolutionary conservation of sRep HS elements and the associated sequence motifs, suggests that those atypical elements play a definite but hitherto unknown function in the viral infective cycle. In absence of any factual data it is only feasible to speculate about the possible function(s) of the sRep HS on the basis of their common characteristics.

Certainly, the most remarkable feature of the sRep HS elements is its complete identity in nucleotide sequence with a specific segment of the rep gene in the cognate DNA-A component, because the evolutionary preservation of such an absolute matching between specific segments of distinct, physically separated DNA molecules, should involve very strong selective pressures against mutations diminishing the identity between the former DNA sequences. Therefore, the function of the sRep HS elements is most likely related to a process that requires a perfect or very high complementarity between DNA and/or RNA molecules, such as the gene regulation by microRNAs (miRNAs).

The miRNAs are ~22-nt-long noncoding RNAs that posttranscriptionally regulate gene expression by binding to specific mRNAs, thus repressing its translation and/or inducing its degradation [55]. Several DNA viruses (i.e., herpesviruses, adenoviruses, ascoviruses and polyomaviruses) encode miRNAs which participate in the regulation of some processes of the viral infection cycle [56, 57]. For example, the simian virus 40 (SV40) encodes a single miRNA which lie antisense to the viral mRNA encoding the T-antigen, a multifunctional protein essential for virus replication. This miRNA is expressed late in infection, hence promoting the T-antigen mRNA degradation and downregulating the synthesis of this protein at late stages of the SV40 replication cycle [58]. In close analogy with SV40 miRNA, the sRep HS elements of begomoviruses are single, discrete noncoding DNA sequences highly similar to a specific segment of the gene encoding the viral replication protein. Further analogies between those heterologous viral sequences are the following: (1) The genomic location of the miRNA, but not its nucleotide sequence, is conserved among polyomaviruses (i.e., SV40, Merkel cell virus, human BK virus, JC virus, and mouse polyomavirus) [5961]; similarly, the location of sRep HS elements within the DNA-B intergenic region, but not its specific sequence, is conserved among begomoviruses (data from this study); (2) The temporal expression of the SV40 miRNA, that is restricted to the late stage of infection, is similar among all the examined polyomaviruses [57, 59]; likewise, although the temporal expression of begomovirus transcripts including the sRep HS region (if any) is unknown, it is plausible than them would be late expressed, because the hypothetical promoter that lead its transcription is similar to begomovirus CP promoters, which are typically active at the late phase of the viral infection cycle [1, 36]; (3) Like the polyomavirus pre-miRNAs, the DNA-B sequences encompassing sRep HS and the neighboring sequences, have the potential to form extensive hairpin structures susceptible to cleavage by RNase III enzymes (i.e., Drosha and Dicer) involved in the processing of pre-miRNAs (data not shown). Taken together, these lines of indirect evidence suggest a potential function of the sRep HS elements in the posttranscriptional regulation of Rep expression, a hypothesis that must be experimentally examined.

Conclusions

The evidence gathered in this study indicates that EuMV-YP and EuMV-Jal, which are members from the strains A and B of Euphorbia mosaic virus respectively, are actually incompatible in replication, hence implying that these viruses probably represent distinct replicating lineages in natural ecosystems. The scenario we propose for the origin of the EuMV A-strain viruses involves a recombination event that substituted the DNA-A core replication module of an EuMV B- strain virus, with the analogous genomic region of a virus related to CpGMV. This intermolecular exchange suddenly changed the replication specificity of the recombinant DNA-A, thus triggering the process that led to the evolutionary differentiation of EuMV into two distinct strains. The fact that more than 30 recognized begomovirus species include two or more classes of viruses with distinct putative RSDs (i.e., analogous to the EuMV strains) suggests that intermolecular recombination events that involve the virion-strand origin of replication and the first part of the rep gene, are quite common in this group of ssDNA viruses, as has been previously pointed out (51, 52, 53). Another relevant result from this study is the discovery of atypical sequences within the intergenic region of the DNA-B component from some NW-begomoviruses, mostly related to EuMV. These sequences include short fragments of the cognate Rep gene located downstream from a potential internal promoter very similar in modular organization to CP promoters of viruses of the SLCV clade. Even though we do not know the actual function of these sRep HS elements, several lines of indirect evidence suggest their participation in the posttranscriptional regulation of Rep expression, an intriguing possibility that is currently being examined in our laboratory.

Methods

Plant samples and DNA extraction

Samples of symptomatic plants exhibiting leaf curling, yellow or golden mosaic, vein chlorosis and/or stunted growth were collected in several farm fields in the State of Jalisco, Mexico, during 2005. Young leaves from symptomatic pepper plants, as well as a variety of weed plants found as underbrush within the field were gathered. Total nucleic acids were extracted of field samples using a modified version of the Dellaporta method [62].

PCR-based detection of begomoviruses

Total DNA extracts from 63 symptomatic plants were used as templates in PCR reactions with degenerate primers designed to amplify two overlapping genomic segments encompassing either the complete DNA-A or DNA-B of begomoviruses belonging to the SLCV clade [63]. The primers SL2150-for (GACGGCRTTGGYGTCTTTGGC) and cpYMAC-rev (TTWGASGCATGNGTACATGCCA) were used to amplify a DNA-A segment encompassing the intergenic region and part of both Rep and CP genes, whereas the primers CP70-for (GGTTGTGAAGGNCCNTGTAAGGTYCA) and SL2150-rev (GCWGCAAAGACACCAAYGCCGT) were utilized to amplify a complementary and partially overlapped DNA-A segment. Amplification of DNA-B sequences was performed with degenerated primers BC1-290-for (GAARTAGTGGAGATCTATGTTRCAYCT) and BV1- 470-rev (CCATGRCTRTGRATYCTWGCRCC), designed to amplify the complete intergenic region together with a part of both the BC1 and BV1 genes. To amplify the remaining part of DNA-B the degenerate primers BC1- 290-rev (CCSATMAGRTGYAACATAGATCTCC) and BV1-310-for (AGGWACRGTNAARATYGARCGTGT) were used. The PCR-products were cloned into the pGEM-T easy vector (Promega) and subjected to Restriction Fragment Length Polymorphism (RFLP)-analysis by using Eco R I in combination with either Hinf I or Msp I. The produced DNA molecules were fractioned in 2.5% agarose gels, and PCR clones with different restriction patterns were sequenced.

Generation of infectious EuMV clones

To clone the full-length genomic components A and B of EuMV-Jal, the DNA extract of one pepper plant infected with both EuMV-Jal and PHYVV collected at the Castillo locality (see Results) was subjected to rolling circle amplification (RCA) using the TempliPhi kit (GE Healthcare, USA) according to the manufacturer's instructions. The full-length EuMV DNA-A was obtained by cutting the RCA-amplified DNA with Xho I and subsequent cloning of the 2.6 Kb DNA molecule into a plasmid vector. The full-length EuMV DNA-B could not be obtained by a similar procedure after several attempts. Consequently, abutted divergent primers designed over the unique BamHI site in the DNA-B of EuMV-Jal were used in a standard PCR procedure, and the generated 2.6 Kb amplicon was cloned into a pGEM-Teasy vector (Promega). The infectious clones of EuMV-Jal A and EuMV-Jal B were generated as follows: the 0.8-Kb BamHI- XbaI fragment of EuMV-A containing the origin of replication was cloned into the BamHI- XbaI sites of a modified pBlueScript plasmid to create pEu-oriA. Subsequently, a full-length DNA-A of EuMV digested with XbaI was inserted into the XbaI site of pEu-oriA to generate the viral replicon pEuMV1.33A. The infectious clone of EuMV-B was generated by an analogous procedure: the full-length DNA-B cloned into pGEM-T easy was digested with NcoI and re-ligated. This procedure deleted a portion of the viral genome, leaving intact all elements important for replication (1.3-kb), thus generating the pEu-oriB plasmid. Finally, a full-length DNA-B digested with BamHI was cloned into the BamH1 site of pEu-oriB, yielding the infectious clone pEuMV1.5B.

Plant infection assays

Nicotiana benthamiana, Capsicum annuum and Datura stramonium plants were inoculated using a low-pressure biolistic method [64]. The target leaves (third- to four-leaf stage) were either directly shot at 100 to 120 psi helium pressure with tungsten particles (0.7 mm, BioRad, Hercules, CA) covered with the EuMV-A and EuMV-B viral DNAs (5 μg), or inoculated mechanically by using carborundum according to a procedure recently described [65]. The inoculated plants were maintained in an insect-free growth chamber (27°C, daily cycle of 16 h light -8 h dark), and subsequently scored for the appearance of disease symptoms. The infection status of the inoculated plants was assessed by visual inspection of symptoms and by PCR analysis of all plants at the end of the experiment.

Reassortment experiments

Pseudorecombination experiments were carried out by biolistically inoculating seedlings of N. benthamiana plants with all possible pair combinations of A and B component clones of both EuMV-Jal and EuMV-YP. Infectious clones of EuMV-Jal were partial tandem repeats of either DNA-A or DNA-B components, as mentioned above, whereas in the case of EuMV-YP, cloned monomeric components were used as previously described [20]. A total of 18 seedlings (three independent experiments, six plants each replicate) were inoculated with each one of the four possible combinations of EuMV-Jal and EuMV-YP genomic components. Mock-inoculated negative controls were included for each replicate. The inoculated plants were maintained in a growth chamber (27°C, daily cycle of 16 h light -8 h dark) and scored for the appearance and development of disease symptoms during 4-5 weeks. All plants, both symptomless and symptomatic, were tested for the presence of viral DNA in newly emerged leaves at 14 dpi by PCR-based detection, using both DNA-A and DNA-B specific primers. Asymptomatic plants were re-examined by PCR at 28 dpi, to detect cases of delayed infection.

Phylogenetic analysis

Full DNA-A and DNA-B sequences from EuMV-Jal were compared with other New World and Old World begomoviruses available at the GenBank-NCBI database, using BLAST-N. The positions and sizes of EuMV-Jal open reading frames were predicted using EditSeq (DNASTAR Inc., Madison, WI). Paired alignments were obtained by the ClustalV and ClustalW methods in the MegAlign application of the Lasergene package (DNASTAR), using the default parameters. Neighbour-joining phylogenetic trees for EuMV DNA-A and DNA-B components were constructed using Mega 4.0 [66] with1,000 bootstrap replicates and pairwise evolutionary distances calculated with a maximum likelihood nucleotide substitution model. Trees were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

Recombination analysis

Detection of potential recombination breakpoints and recombinant sequences was carried out using the suite of recombination detection methods implemented in RDP software (29). A sequence alignment containing four EuMV isolates and closely related species (sharing >76% of nucleotide identity with EuMV-Jal) was used as input data for RDP. The analysis was performed using the default settings in all detection methods, with a Bonferroni corrected P-value cut-off of 0.05.

Notes

Declarations

Acknowledgements

We thank Salvador Ambriz-Granados for his technical assistance, and Dr. Braulio Gutierrez-Medina for critical reading of the manuscript. JGJ, ABA and BBH were supported by a PhD fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACYT, Mexico). This research was partially supported by a CONACYT grant (SEP-84004) to GAA.

Authors’ Affiliations

(1)
Instituto Potosino de Investigación Científica y Tecnológica, A.C.,Camino a la Presa San José,SLP
(2)
Universidad Autónoma Agraria Antonio Narro, Departamento de Parasitología Agrícola.Bellavista
(3)
3Centro de Investigación Científica de Yucatán

References

  1. Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D: Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit Rev Plant Sci 1999, 18: 71-106. 10.1016/S0735-2689(99)00383-4View ArticleGoogle Scholar
  2. Zhang W, Olson NH, Baker TS, Faulkner L, Agbandje-McKenna M, Boulton MI, Davies JW, McKenna R: Structure of the Maize streak virus geminate particle. Virology 2001, 279: 471-477. 10.1006/viro.2000.0739PubMedView ArticleGoogle Scholar
  3. Stanley J, Bisaro DM, Briddon RW, Brown JK, Fauquet CM, Harrison BD, Rybicki EP, Stenger DC: Geminiviridae. In Virus Taxonomy (VIIIth Report of the ICTV). Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. Elsevier/Academic Press, London; 2005:301-306.Google Scholar
  4. Rojas MR, Hagen C, Lucas WJ, Gilbertson RL: Exploiting chinks in the plant's armor: evolution and emergence of geminiviruses. Annu Rev Phytopathol 2005, 43: 361-394. 10.1146/annurev.phyto.43.040204.135939PubMedView ArticleGoogle Scholar
  5. Seal SE, vandenBosch F, Jeger MJ: Factors influencing begomovirus evolution and their increasing global significance: implications for sustainable control. Crit Rev Plant Sci 2006, 25: 23-46. 10.1080/07352680500365257View ArticleGoogle Scholar
  6. Briddon RW, Patil BL, Bagewadi B, Nawaz-ul-Rehman MS, Fauquet CM: Distinct evolutionary histories of the DNA-A and DNA-B components of bipartite begomoviruses. BMC Evol Biol 2010, 10: 97. 10.1186/1471-2148-10-97PubMedPubMed CentralView ArticleGoogle Scholar
  7. Fauquet CM, Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou X: Geminivirus strain demarcation and nomenclature. Arch Virol 2008, 153: 783-821. 10.1007/s00705-008-0037-6PubMedView ArticleGoogle Scholar
  8. Bisaro DM: Silencing suppression by geminivirus proteins. Virology 2006, 344: 158-68. 10.1016/j.virol.2005.09.041PubMedView ArticleGoogle Scholar
  9. Hehnle S, Wege C, Jeske H: Interaction of DNA with the movement proteins of geminiviruses revisited. J Virol 2004, 78: 698-706. 10.1128/JVI.78.14.7698-7706.2004View ArticleGoogle Scholar
  10. Sanderfoot AA, Lazarowitz SG: Getting it together in plant virus movement: cooperative interactions between bipartite geminivirus movement proteins. Trends Cell Biol 1996, 9: 353-358. 10.1016/0962-8924(96)10031-3View ArticleGoogle Scholar
  11. Fontes EP, Gladfelter HJ, Schaffer RL, Petty IT, Hanley-Bowdoin L: Geminivirus replication origins have a modular organization. Plant Cell 1994, 6: 405-416. 10.1105/tpc.6.3.405PubMedPubMed CentralView ArticleGoogle Scholar
  12. Singh DK, Malik PS, Choudhury NR, Mukherjee SK: MYMIV replication initiator protein (Rep): roles at the initiation and elongation steps of MYMIV DNA replication. Virology 2008, 380: 75-83. 10.1016/j.virol.2008.07.010PubMedView ArticleGoogle Scholar
  13. Rojas A, Kvarnheden A, Marcenaro D, Valkonen JP: Sequence characterization of tomato leaf curl Sinaloa virus and tomato severe leaf curl virus: phylogeny of New World begomoviruses and detection of recombination. Arch Virol 2005, 150: 1281-1299. 10.1007/s00705-005-0509-xPubMedView ArticleGoogle Scholar
  14. Rybicki EP: A phylogenetic and evolutionary justification for 3 genera of Geminiviridae . Arch Virol 1994, 139: 49-77. 10.1007/BF01309454PubMedView ArticleGoogle Scholar
  15. Argüello-Astorga G, Herrera-Estrella L, Rivera-Bustamante R: Experimental and theoretical definition of geminivirus origin of replication. Plant Mol Biol 1994, 26: 553-556. 10.1007/BF00013742PubMedView ArticleGoogle Scholar
  16. Argüello-Astorga GR, Guevara-González RG, Herrera-Estrella LR, Rivera-Bustamante RF: Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication. Virology 1994, 203: 90-100. 10.1006/viro.1994.1458PubMedView ArticleGoogle Scholar
  17. Argüello-Astorga GR, Ruiz-Medrano R: An iteron-related domain is associated to motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach. Arch Virol 1994, 146: 1465-1485. 10.1007/s007050170072View ArticleGoogle Scholar
  18. Bird J, Sánchez J, Rodríguez RL, Juliá FJ: Rugaceous (whitefly-transmitted) viruses in Puerto Rico. In Tropical Diseases of Legumes. Edited by: Bird J, Maramorosch K. New York, USA: Academic Press; 1975:3-25.Google Scholar
  19. Costa AS: Whitefly-transmitted plant diseases. Annu Rev Phytopathol 1976, 14: 429-49. 10.1146/annurev.py.14.090176.002241View ArticleGoogle Scholar
  20. Hernández-Zepeda C, Idris AM, Carnevali G, Brown JK, Moreno-Valenzuela OA: Molecular characterization and experimental host range of Euphorbia mosaic virus-Yucatan Peninsula, a begomovirus species in the Squash leaf curl virus clade. Plant Pathol 2007, 56: 763-770. 10.1111/j.1365-3059.2007.01652.xView ArticleGoogle Scholar
  21. Collins AM, Brown JK, Rehman MM, Roye ME: Complete nucleotide sequence of an isolate of Euphorbia mosaic virus that infects Euphorbia heterophylla and Wissadula amplissima in Jamaica. Arch Virol 2009, 154: 1859-1860. 10.1007/s00705-009-0508-4PubMedPubMed CentralView ArticleGoogle Scholar
  22. Méndez-Lozano J, Torres-Pacheco I, Fauquet CM, Rivera-Bustamante RF: Interactions between geminiviruses in a naturally occurring mixture: Pepper huasteco virus and Pepper golden mosaic virus . Phytopathology 2003, 93: 270-277. 10.1094/PHYTO.2003.93.3.270PubMedView ArticleGoogle Scholar
  23. Torres-Pacheco I, Garzón-Tiznado JA, Brown JK, Becerra-Flora A, Rivera-Bustamante RF: Detection and distribution of Geminiviruses in Mexico and the Southern United States. Phytopathology 1996, 86: 1186-1192. 10.1094/Phyto-86-1186View ArticleGoogle Scholar
  24. Torres-Pacheco I, Garzón-Tiznado JA, Herrera-Estrella L, Rivera-Bustamante RF: Complete nucleotide sequence of pepper huasteco virus: analysis and comparison with bipartite geminiviruses. J Gen Virol 1993, 74: 2225-2231. 10.1099/0022-1317-74-10-2225PubMedView ArticleGoogle Scholar
  25. Shepherd DN, Martin DP, Lefeuvre P, Monjane AL, Owor BE, Rybicki EP, Varsani A: A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. J Virol Methods 2008, 149: 97-102. 10.1016/j.jviromet.2007.12.014PubMedView ArticleGoogle Scholar
  26. Hernández-Zepeda C, Argüello-Astorga G, Idris AM, Carnevali G, Brown JK, Moreno-Valenzuela OA: Molecular characterization and phylogenetic relationships of Desmodium leaf distortion virus (DeLDV): a new begomovirus infecting Desmodium glabrum in Yucatan, Mexico. Virus Genes 2009, 39: 371-374. 10.1007/s11262-009-0398-2PubMedView ArticleGoogle Scholar
  27. García-Andrés S, Tomás DM, Sánchez-Campos S, Navas-Castillo J, Moriones E: Frequent occurrence of recombinants in mixed infections of tomato yellow leaf curl disease-associated begomoviruses. Virology 2007, 365: 210-219. 10.1016/j.virol.2007.03.045PubMedView ArticleGoogle Scholar
  28. van der Walt E, Rybicki EP, Varsani A, Polston JE, Billharz R, Donaldson L, Monjane AL, Martin DP: Rapid host adaptation by extensive recombination. J Gen Virol 2009, 90: 734-746. 10.1099/vir.0.007724-0PubMedPubMed CentralView ArticleGoogle Scholar
  29. Martin DP, Williamson C, Posada D: RDP2: recombination detection and analysis from sequence alignments. Bioinformatics 2005, 21: 260-262. 10.1093/bioinformatics/bth490PubMedView ArticleGoogle Scholar
  30. Ilyina TV, Koonin EV: Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res 1992, 20: 3279-3285. 10.1093/nar/20.13.3279PubMedPubMed CentralView ArticleGoogle Scholar
  31. Argüello-Astorga G, Herrera-Estrella L: Ancestral multipartite units in light-responsive plant promoters have structural features correlating with specific phototransduction pathways. Plant Physiol 1996, 112: 1151-1166. 10.1104/pp.112.3.1151PubMedPubMed CentralView ArticleGoogle Scholar
  32. Argüello-Astorga G, Herrera-Estrella L: Evolution of light-regulated plant promoters. Annu Rev Plant Physiol Plant Mol Biol 1998, 49: 525-555. 10.1146/annurev.arplant.49.1.525PubMedView ArticleGoogle Scholar
  33. Hernandez-Zepeda C, Brown JK, Moreno-Valenzuela OA, Arguello-Astorga G, Idris AM, Carnevali G, Rivera-Bustamante RF: Characterization of Rhynchosia yellow mosaic Yucatan virus , a new recombinant begomovirus associated with two fabaceous weeds in Yucatan, Mexico. Arch Virol 2010, 155: 1571-1579. 10.1007/s00705-010-0730-0PubMedView ArticleGoogle Scholar
  34. Lescot M, Déhais P, Moreau Y, De Moor B, Rouzé P, Rombauts S: PlantCARE: a database of plant cis -acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 2002, 30: 325-327. 10.1093/nar/30.1.325PubMedPubMed CentralView ArticleGoogle Scholar
  35. Higo K, Ugawa Y, Iwamoto M, Korenaga T: Plant cis -acting regulatory DNA elements (PLACE) database:1999. Nucleic Acids Research 1999, 27: 297-300. 10.1093/nar/27.1.297PubMedPubMed CentralView ArticleGoogle Scholar
  36. Ruiz-Medrano R, Guevara-Gonzalez RG, Arguello-Astorga GR, Monsalve-Fonnegra Z, Herrera-Estrella LR, Rivera-Bustamante RF: Identification of a sequence element involved in AC2-mediated transactivation of the Pepper huasteco virus coat protein gene. Virology 1999, 253: 162-169. 10.1006/viro.1998.9484PubMedView ArticleGoogle Scholar
  37. Itzhaki H, Maxson JM, Woodson WR: An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S transferase (GST1) gene. Proc Natl Acad Sci USA 1994, 91: 8925-8929. 10.1073/pnas.91.19.8925PubMedPubMed CentralView ArticleGoogle Scholar
  38. Montgomery J, Goldman S, Deikman J, Margossian L, Fischer RL: Identification of an ethylene responsive region in the promoter of a fruit ripening gene. Proc Natl Acad Sci USA 1993, 90: 5939-5943. 10.1073/pnas.90.13.5939PubMedPubMed CentralView ArticleGoogle Scholar
  39. Rawat R, Xu Z-F, Yao K-M, Chye ML: Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase. Plant Mol Biol 2005, 57: 629-643. 10.1007/s11103-005-0954-7PubMedView ArticleGoogle Scholar
  40. Petty ITD, Coutts RHA, Buck KW: Transcriptional mapping of the coat protein gene of tomato golden mosaic virus. J Gen Virol 1988, 69: 1359-1365. 10.1099/0022-1317-69-6-1359View ArticleGoogle Scholar
  41. Paprotka T, Metzler V, Jeske H: The first DNA 1-like alpha satellites in association with New World begomoviruses in natural infections. Virology 2010, 404: 148-157. 10.1016/j.virol.2010.05.003PubMedView ArticleGoogle Scholar
  42. Hanson SF, Hoogstraten RA, Ahlquist P, Gilbertson RL, Russell DR, Maxwell DP: Mutational analysis of a putative NTP-binding domain in the replication-associated protein (AC1) of Bean golden mosaic geminivirus. Virology 1995, 211: 1-9. 10.1006/viro.1995.1373PubMedView ArticleGoogle Scholar
  43. Koonin EugeneV: A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res 1993, 21: 2541-2547. 10.1093/nar/21.11.2541PubMedPubMed CentralView ArticleGoogle Scholar
  44. Cazzonelli CI, Burke J, Velten J: Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco. Plant Mol Biol 2005, 58: 465-81. 10.1007/s11103-005-6589-xPubMedView ArticleGoogle Scholar
  45. Velten J, Morey KJ, Cazzonelli CI: Plant viral intergenic DNA sequence repeats with transcription enhancing activity. Virol J 2005, 2: 16-24. 10.1186/1743-422X-2-16PubMedPubMed CentralView ArticleGoogle Scholar
  46. Eagle PA, Hanley-Bowdoin L: Cis elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication. J Virol 1997, 71: 6947-6955.PubMedPubMed CentralGoogle Scholar
  47. Gibbs AJ, Gibbs MJ: A broader definition of the virus species. Arch Virol 2006, 151: 1419-1422. 10.1007/s00705-006-0775-2PubMedView ArticleGoogle Scholar
  48. Van Regenmortel MH: Applying the species concept to plant viruses. Arch Virol 1989, 104: 1-17. 10.1007/BF01313804PubMedView ArticleGoogle Scholar
  49. Chatterji A, Padidam M, Beachy RN, Fauquet CM: Identification of replication specificity determinance in tomato leaf curl virus from New Delhi. J Virol 1999, 73: 5481-5489.PubMedPubMed CentralGoogle Scholar
  50. Padidam M, Beachy RN, Fauquet CM: Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity. J Gen Virol 1995, 76: 25-35. 10.1099/0022-1317-76-1-25PubMedView ArticleGoogle Scholar
  51. Lefeuvre P, Lett JM, Varsani A, Martin DP: Widely conserved recombination patterns among single-stranded DNA viruses. J Virol 2009, 83: 2697-707. 10.1128/JVI.02152-08PubMedPubMed CentralView ArticleGoogle Scholar
  52. Lefeuvre P, Lett JM, Reynaud B, Martin DP: Avoidance of protein fold disruption in natural virus recombinants. PLoS Pathog 2007, 3: e181. 10.1371/journal.ppat.0030181PubMedPubMed CentralView ArticleGoogle Scholar
  53. Varsani A, Shepherd DN, Monjane AL, Owor BE, Erdmann JB, Rybicki EP, Peterschmitt M, Briddon RW, Markham PG, Oluwafemi S, Windram OP, Lefeuvre P, Lett JM, Martin DP: Recombination, decreased host specificity and increased mobility may have driven the emergence of maize streak virus as an agricultural pathogen. J Gen Virol 2008, 89: 2063-2074. 10.1099/vir.0.2008/003590-0PubMedPubMed CentralView ArticleGoogle Scholar
  54. Londoño A, Riego-Ruiz L, Arguello-Astorga GR: DNA-binding specificity determinants of replication proteins encoded by eukaryotic ssDNA viruses are adjacent to widely separated RCR conserved motifs. Arch Virol 2010, 155: 1033-1046. 10.1007/s00705-010-0674-4PubMedView ArticleGoogle Scholar
  55. Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116: 281-297. 10.1016/S0092-8674(04)00045-5PubMedView ArticleGoogle Scholar
  56. Nair V, Zavolan M: Virus-encoded microRNAs: novel regulators of gene expression. Trends Microbiol 2006, 14: 169-175. 10.1016/j.tim.2006.02.007PubMedView ArticleGoogle Scholar
  57. Skalsky RL, Cullen BR: Viruses, microRNAs, and host interactions. Annu Rev Microbiol 2010, 64: 123-141. 10.1146/annurev.micro.112408.134243PubMedPubMed CentralView ArticleGoogle Scholar
  58. Sullivan CS, Grundhoff AT, Tevethia S, Pipas JM, Ganem D: SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 2005, 435: 682-686. 10.1038/nature03576PubMedView ArticleGoogle Scholar
  59. Cullen BR: Five Questions about Viruses and MicroRNAs. PLoS Pathog 2010, 6: e1000787. 10.1371/journal.ppat.1000787PubMedPubMed CentralView ArticleGoogle Scholar
  60. Seo GJ, Fink LH, O'Hara B, Atwood WJ, Sullivan CS: Evolutionarily conserved function of a viral microRNA. J Virol 2008, 82: 9823-9828. 10.1128/JVI.01144-08PubMedPubMed CentralView ArticleGoogle Scholar
  61. Sullivan CS, Sung CK, Pack CD, Grundhoff A, Lukacher AE: Murine Polyomavirus encodes a microRNA that cleaves early RNA transcripts but is not essential for experimental infection. Virology 2009, 387: 157-167. 10.1016/j.virol.2009.02.017PubMedPubMed CentralView ArticleGoogle Scholar
  62. Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: version II. Plant Mol Biol Rep 1983, 1: 19-21. 10.1007/BF02712670View ArticleGoogle Scholar
  63. Mauricio-Castillo JA, Argüello-Astorga GR, Alpuche-Solís AG, Monreal-Vargas CT, Díaz-Gómez O, de la Torre-Almaraz R: First Report of Tomato severe leaf curl virus in México. Plant Dis 2006, 90: 1116. 10.1094/PD-90-1116AView ArticleGoogle Scholar
  64. Carrillo-Tripp J, Lozoya-Gloria E, Rivera-Bustamante RF: Symptom remission and specific resistance of pepper plants after infection by Pepper golden mosaic virus . Phytopathology 2007, 97: 51-57. 10.1094/PHYTO-97-0051PubMedView ArticleGoogle Scholar
  65. Ascencio-Ibañez JT, Settlage SB: DNA abrasion onto plants is an effective method for geminivirus infection and virus induced gene silencing. J Virol Met 2007, 142: 198-203. 10.1016/j.jviromet.2007.01.031View ArticleGoogle Scholar
  66. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24: 1596-1599. 10.1093/molbev/msm092PubMedView ArticleGoogle Scholar
  67. Sunter G, Bisaro DM: Identification of a minimal sequence required for activation of the Tomato golden mosaic virus coat protein promoter in protoplasts. Virology 2003, 305: 452-462. 10.1006/viro.2002.1757PubMedView ArticleGoogle Scholar

Copyright

© Gregorio-Jorge et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement