Transcript mapping of Cotton leaf curl Burewala virusand its cognate betasatellite, Cotton leaf curl Multan betasatellite
© Akbar et al.; licensee BioMed Central Ltd. 2012
Received: 6 June 2012
Accepted: 25 October 2012
Published: 29 October 2012
Whitefly-transmitted geminiviruses (family Geminiviridae, genus Begomovirus) are major limiting factors for the production of numerous dicotyledonous crops throughout the warmer regions of the world. In the Old World a small number of begomoviruses have genomes consisting of two components whereas the majority have single-component genomes. Most of the monopartite begomoviruses associate with satellite DNA molecules, the most important of which are the betasatellites. Cotton leaf curl disease (CLCuD) is one of the major problems for cotton production on the Indian sub-continent. Across Pakistan, CLCuD is currently associated with a single begomovirus (Cotton leaf curl Burewala virus [CLCuBuV]) and the cotton-specific betasatellite Cotton leaf curl Multan betasatellite (CLCuMuB), both of which have recombinant origins. Surprisingly, CLCuBuV lacks C2, one of the genes present in all previously characterized begomoviruses. Virus-specific transcripts have only been mapped for few begomoviruses, including one monopartite begomovirus that does not associate with betasatellites. Similarly, the transcripts of only two betasatellites have been mapped so far. The study described has investigated whether the recombination/mutation events involved in the evolution of CLCuBuV and its associated CLCuMuB have affected their transcription strategies.
The major transcripts of CLCuBuV and its associated betasatellite (CLCuMuB) from infected Nicotiana benthamiana plants have been determined. Two complementary-sense transcripts of ~1.7 and ~0.7 kb were identified for CLCuBuV. The ~1.7 kb transcript appears similar in position and size to that of several begomoviruses and likely directs the translation of C1 and C4 proteins. Both complementary-sense transcripts can potentially direct the translation of C2 and C3 proteins. A single virion-sense transcript of ~1 kb, suitable for translation of the V1 and V2 genes was identified. A predominant complementary-sense transcript was also confirmed for the betasatellite.
Overall, the transcription of CLCuBuV and the recombinant CLCuMuB is equivalent to earlier mapped begomoviruses/betasatellites. The recombination events that featured in the origins of these components had no detectable effects on transcription. The transcripts spanning the mutated C2 gene showed no evidence for involvement of splicing in restoring the ability to express intact C2 protein.
Viruses of the Geminiviridae family have circular single-stranded (ss)DNA genomes and classify into four genera according to their host range, insect vector and genome organization . They are widely distributed throughout the world and infect either monocotyledonous or dicotyledonous hosts. Geminiviruses of the genus Begomovirus are transmitted by the whitefly Bemisia tabaci and have genomes that consist of either a single or two ssDNA components. The two components of bipartite begomoviruses are known as DNA A and DNA B and both are, for most species, essential for symptomatic infection of plants. Monopartite begomoviruses are often associated with DNA satellites known as alphasatellites and betasatellites . The satellite-associated begomoviruses are widespread in the Old World and represent the largest group of begomoviruses, outnumbering both the truly monopartite and the bipartite begomoviruses.
Geminiviruses replicate via a double-stranded (ds) DNA intermediate that is also used as a template for transcription. Transcription is bidirectional to generate mRNAs diverging from an intergenic region. Transcripts initiate downstream of either consensus TATA box motifs or initiator elements, suggesting that host RNA polymerase II transcribes the viral mRNAs. The viral RNAs are polyadenylated and composed of multiple RNA species, indicating the complexity of geminiviral transcription .
The genomes of monopartite begomoviruses encode six genes, two in the virion-sense orientation (V1 and V2) and four in the complementary-sense orientation (C1 to C4). The V1 gene encodes the coat protein (CP), involved in virus movement within and between plants [4, 5], and the V2 protein which is involved in virus movement in plants as well as overcoming RNA silencing host defences triggered by dsRNA (also known as post-transcriptional gene silencing [PTGS]) for some virus species [6, 7]. In the complementary-sense the C1 gene encodes a rolling-circle replication initiator protein (known as the replication-associated protein [Rep]), that also interferes with host cell-cycle and is the only virus encoded protein required for virus replication, the C2 protein (known as the transcriptional-activator protein for some begomoviruses) that is involved in up-regulating late, virion-sense encoded genes (in some cases) as well as host genes and is a RNA silencing suppressor [6, 8–10]. The product of the C3 gene, known as the replication enhancer protein (REn), is involved in virus DNA replication (by interacting with Rep) and also interacts with host components . The product of the C4 gene may be a symptom determinant and also exhibits RNA silencing suppressor activity .
The betasatellites are a recently identified class of ssDNA satellites . They are, in many cases, required by their helper begomoviruses to symptomatically infect the hosts from which they were isolated [12, 13]. Betasatellites encode a dominant symptom/pathogenicity determinant (known as βC1) which is a suppressor of PTGS and may be involved in virus movement in plants and enhance virus DNA levels in plants [6, 14–17].
Cotton leaf curl disease (CLCuD) is the most significant problem for cotton production across most of Pakistan and northwestern areas of India . This disease appeared in epidemic form in 1991–92 and is caused by monopartite begomoviruses (seven distinct species were identified), often as multiple infections , and a specific betasatellite – Cotton leaf curl Multan betasatellite (CLCuMuB). In the late 1990’s the introduction of resistant cotton varieties restored cotton production in Pakistan to pre-epidemic levels. However, from 2001–2002 onwards, the disease appeared on all previously resistant varieties, an indication of the appearance of a strain of the disease with the ability to break resistance (now known as the “Burewala” strain) . CLCuD in resistant cotton varieties has been shown to be associated with a single begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), which has spread across most areas of Pakistan and into India [21–23]. CLCuBuV is a recombinant virus consisting of sequences derived from two parents, Cotton leaf curl Kokhran virus (CLCuKoV; which donated the virion-sense sequences) and Cotton leaf curl Multan virus (CLCuMuV; which donated the complementary-sense sequences). These two species were dominant in cotton prior to resistance breaking [19, 21]. Significantly, CLCuBuV lacks an intact C2 gene. This is surprising since all begomoviruses, curtoviruses and the only known topocurvirus reported to date have a C2 gene that potentially encodes an ~134aa protein . C2 is a multifunctional protein that plays an important role in host-virus interactions.
The betasatellite associated with CLCuBuV was also shown to be recombinant. This consists of the original CLCuMuB with a small fragment (~80 nt), in a non-coding sequence, derived from a betasatellite first identified in tomato [21, 24]. In common with all betasatellites, CLCuMuB encodes a single gene, βC1.
The work presented here consisted of mapping the major transrcripts of CLCuBuV and its associated CLCuMuB for comparison to the transcript maps of other begomoviruses/betasatellites and investigating whether the recombination/mutation events involved in their evolution have affected gene expression at the level of transcription.
Infection of plants and RNA isolation
The begomovirus clones CLCuBuV–[PK:Veh2:4] (accession number AM421522) and associated betasatellite, CLCuMuB-[PK:Veh:06] (AM774307) were used to infect N. benthamiana plants as previously described .
Total RNA was isolated from plants using Trizol reagent (Gibco-BRL) as described by the manufacturer. The extracted RNA was dissolved in diethylpyrocarbonate-treated water and stored at −80°C.
Northern blot hybridization
Oligonucleotide primers used in the study
Oligonucleotide sequence (5′ to 3′)
5′RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) PCR
5′ RLM-RACE PCR was performed using an RLM-RACE Kit (Ambion) according to the manufacturer’s instructions. Total RNA was treated with alkaline phosphatase to remove the 5′ phosphate group of non-capped mRNAs, followed by treatment with tobacco acid pyrophosphatase (TAP) to decap mRNAs. TAP treated RNA was ligated to a synthetic 5′ RNA adapter (Table 1) using T4 RNA Ligase. This RNA was reverse transcribed and PCR was performed with specific primer combinations (Table 1) to amplify 5′ termini of virus and betasatellite specific transcripts.
3′ RLM-RACE PCR was performed according to the manufacturer’s instructions (Ambion). Total RNA (1 μg) was reverse transcribed with an oligo (dT) adapter primer (Table 1), and cDNA was used in PCR with specific primer combinations (Table 1) to amplify 3′ termini of virus and betasatellites specific transcripts.
Cloning and sequencing of RLM-RACE PCR products
PCR products were purified using a gel extraction kit (Fermentas) according to the manufacturer’s instructions, and were cloned into the pTZ57R/T vector (Fermentas). PCR products (without cloning), as well as cloned products, were sequenced (Macrogen, Korea). Sequences were analyzed using the Lasergene software package (DNASTAR Inc.).
Northern blot analysis of virus-specific RNAs in CLCuBuV/CLCuMuB-infected N. benthamianaplants
High-resolution mapping of the CLCuBuV complementary-sense transcripts
High-resolution mapping of the CLCuBuV virion-sense transcripts
The 3′ RACE outer primer was used in combination with the gene specific primer v2cpb1 in the primary PCR and then nested PCR was performed using the 3′RACE inner primer and gene specific primer v2cp-′3b to determine the 3′ ends of virion-sense transcripts. The transcripts spanning genes V1 and V2 had the same predominant 3′ end with the transcription termination site mapping at nt 1083 and 3′ untranslated regions (UTRs) of 21nt (Figure 4).
Mapping the 3′ and 5′ends of βC1 transcript of CLCuMuB
3′ RLM-RACE clones showed transcription termination mapping at coordinate 190 (Figure 6). Gene specific primers BC1-3′ (outer) and BC2bb (inner) were used in combination with 3′ RACE outer and inner primers respectively (Table 1).
Although the transcription strategies of a few begomoviruses have been determined [25–29], including one monopartite begomovirus (Tomato leaf curl virus [ToLCV]), none of the betasatellite-associated begomoviruses are amongst these. Only for two betasatellites, Ageratum yellow vein betasatellite (AYVB) and the non-recombinant CLCuMuB [15, 30], have transcripts been determined. It was therefore of interest to determine the transcripts of CLCuBuV, and its associated recombinant betasatellite. Particular attention was paid to transcription across the mutation affecting the CLCuBuV C2 gene to determine whether any transcriptional changes resulted from the mutation.
In common with other geminiviruses CLCuBuV produces multiple overlapping polycistronic RNA species that diverge from the IR, confirming a bidirectional transcription strategy. Transcription of the complementary-sense is more complex, producing multiple RNAs species with distinct 5′ ends and a common 3′ end. The polyadenylation sites are arranged such that complementary- and virion-sense RNAs overlap at their 3′ ends [29, 31]. Though complementary and virion-sense transcripts overlap only by a small region, this unusual read-through transcription on a circular viral DNA could produce longer transcripts that are complementary, forming dsRNA, that may act to trigger RNA silencing [29, 32]. Hybridization smears (Figure 1) detected on northern blots may represent the breakdown products of RNA formed from such aberrant read-through transcription. The additional hybridization signals detected on northern blots may represent short ORFs (Figure 1 panel, B). Such additional signals representing short ORFs have been mapped for the BC1 transcription unit of Mungbean yellow mosaic virus (MYMV). These short ORFs may inhibit the translation of downstream genes unless leaky scanning occurs. Leaky scanning occurs when the 5′ most initiation codon has a weak context and ribosomes instead initiate translation at a downstream codon .
The virion-sense transcripts of CLCuBuV are comparable in size and location to those of other begomoviruses and the curtovirus Spinach curly top virus (SpCTV)[28, 31]. Mapping of the virion-sense transcripts of CLCuBuV identified two major 5′ termini. One 5′ end mapped upstream of the V1 and V2 genes and could potentially direct the translation of both of these genes, whereas the second 5′ end mapped upstream of V1, and could potentially serve to translate only the CP (Figure 5). The transcript spanning the V1, with 5′ end mapped at coordinate 147, has an untranslated leader of 145 nt. An untranslated leader of ~160 nt has previously been reported for the V1 gene of ACMV . Analysis of the sequences of virion-sense transcripts of CLCuBuV indicated the presence of TATA box sequences at 34 and 49 nt upstream of the V2 start methionine codon. The size of RNAs characterised here is consistent with the ~1 kb virion-sense RNA detected in infected tissue (Figure 1, panel B).
The analysis detected no RNAs that span only the C3, suggesting that REn is expressed only from polycistronic mRNAs. The transcripts spanning the C2 and C3 genes are of two classes, short (initiating at coordinates 1628 and 1636) and long (initiating at 1752)(Figure 2).The two transcripts have short 5′ UTRs of 20 and 28 nt, whereas the long transcript has a longer 5′ UTR of 144 nt (Figure 2). Tomato golden mosaic virus (TGMV) also transcribes to give two size classes of transcripts encompassing C2 and C3. For TGMV there are additional AUG initiation codons in the 5′ UTR of the longer C2/C3 transcript. The first of these has the capacity to translate the C-terminal 122aa of the Rep, terminating after the C2 initiation codon, was shown to be inhibitory for C2 and REn expression . For CLCuBuV there is a single AUG initiation codon in the 5′ UTR of the 1752 transcript (Figure 2), out of frame with the C1, which terminates 11 aa before the C2 AUG initiation codon. This is unlikely to significantly affect C2 translation since the additional AUG initiation codon in the 5′ UTR is in an unfavourable context (tAtAAUGaaU) compared to the consensus context for dicot mRNAs (aaA[A/C]aAUGGCu), whereas both the C2 and C3 AUG initiation codons are in a more favourable context (AAAAAAUGca and cAACcAUGGa). Thus in contrast to TGMV, CLCuBuV may translate both C2 and REn from both the size classes of C2/C3 transcripts.
The mechanism by which REn is expressed is unclear. The gap between both the C1 and (truncated) C2 terminator codons and the start of C3 suggests that it may involve reinitiation or internal initiation. For TGMV the translation of C3 from the short transcript may occur by leaky scanning, since the C3 initiation codon occurs before the C2 terminator codon . However, for CLCuBuV the frame-shift mutation has resulted in the C2 termination codon occurring before the C3 initiation codon and there is an ORF (indicated as C* in Figure 2; this ORF would not encode C2 amino acid sequences), in-frame with C1 and immediately after the C1 termination codon), which could possibly be expressed by leaky scanning through the C2 initiation codon. Translation of the C* ORF might adversely affect C3 expression. However, further studies will be required to investigate these possibilities.
It remains unclear why geminiviruses would express REn from multiple transcripts. A possible explanation is that this protein may be required in larger amounts than possible from a single transcript or its expression requires more subtle control. The protein may be required at several stages of the virus infection cycle and thus may be expressed at different times from different mRNAs.
The transcript 5′ ends identified represent heterogeneous initiation sites of a bicistronic mRNA encoding both C2 and C3. The predicted sizes of RNAs initiating at the 5′ and 3′ends mapped here is consistent with the complementary-sense RNAs detected in infected tissue. Identified transcription start sites are located downstream of putative TATA boxes at optimal distances of 20 to 35 nt. These results suggest that CLCuBuV uses a bicistronic transcription strategy, in common with other geminiviruses, to translate C2 and REn from a single transcript . This is supported by the fact that C3 gene-specific primers did not reveal any major transcription start between the C2 and C3 start codons, or splicing to remove the upstream C2 start codon.
For the long complementary-sense transcripts TATA and CAAT boxes are located at the requisite distances of approximately 30bp from the transcription initiation sites (Figure 3). The longer 1.7 kb transcript appears to use the invariant nonanucleotide sequence on the complementary strand as the promoter element (TATA box). Interestingly, the longest transcript maps at nt 66 with a 5′ UTR of 143 nt that spans the origin of replication and promoter elements far from the complementary-sense genes. A similar transcript has previously been observed for TGMV . For CLCuBuV the short 5′ UTRs are 20–40 nt long and the promoter elements are located at an optimal distance from transcription initiation sites as reported previously for others geminiviruses and thus represent the authentic 5′ ends for these transcripts . For the longer 5′ UTRs, these elements are far removed from the transcription initiation sites. It has been recognized that translation initiation efficiency from the start codon (AUG) depends on its position and sequence context within, and also on its distance from, the 5′ UTR . Short 5′ UTRs may be associated with a decrease in translation initiation efficiency and vice versa .
The RACE clone sequences for the complementary-sense showed a single predominant 3′ end located between the converging V1 and C3 genes. Transcription termination occurs immediately following the stop codon for the C3 gene. These transcripts do not have 3′ UTRs - polyadenylation occurs immediately following the stop codon of the C3 gene. The poly (A) tail of the mRNA is added 21 nt downstream of canonical AATAAA polyadenylation signal. This site is located at an optimal distance downstream of a consensus polyadenylation termination signal suggesting that it represents the authentic 3′ end for these transcripts. Polyadenylation signals normally occur 10–30 nt upstream of the polyadenalytion site . The 3′ UTR for virion-sense transcripts determined in this study are short, being 21 nt. The poly (A) tail is added 22 nt downstream of a polyadenylation signal. A similar arrangement has been observed for other geminiviruses; MYMV and SpCTV [29, 31].
The single transcript identified for CLCuMuB maps with 5′ and 3′ ends at coordinates 563 and 190, respectively (Figure 6). These results are in agreement with the previous transcript mapping of the non-recombinant CLCuMuB . The transcript is polyadenylated and the polyadenylation signal is 18 nt upstream of the stop codon. The putative polyadenylation signal of the non-recombinant CLCuMuB (AAATAA) differs from that of recombinant CLCuMuB (GAATAA). In contrast the putative TATA box element is the same for both CLCuMuB isolates, being 43 nt upstream of the start codon and 31 nt upstream of transcription start site. These results support the predicted TATA box and start codon for βC1 and suggest that transcription may be initiated from a consensus TATA box sequence at an optimal distance of 20–35 nt. The 5′ UTR identified here is of 12 nt long and a transcript with a short leader sequence of eight nucleotides has previously been observed for AYVB . The critical length of leader sequences is 7 nucleotides . The differences between the results obtained here and those of previously obtained for AYVB  (multiple widely spaced 3′ termini and some minor 5′ termini) and for the non-recombinant CLCuMuB (one additional minor 3′ and 5′ terminus) , may be due to differences in experimental approach. For both previous analyses of βC1 transcripts, RNA was extracted from transgenic plants harbouring dimeric betasatellite constructs – such plants would not be expected to contain episomally replicating satellite. The transcripts here are from a bona fide begomovirus-betasatellite infection. However, Saunders et al.  attributed some of the diversity in 3′ termini to the presence of a cryptic polyadenylation in the sequence of AYVB. No such cryptic polyadenylation signals (although the sequence of the presumed polyadenylation signal differs from the consensus, as mentioned earlier) are present in the recombinant CLCuMuB.
Sequencing of clones of the short transcript spanning the C2 mutation in CLCuBuV did not show any splicing, or other editing event, which might restore expression of a full-length C2 protein, supporting the conclusion of Amrao et al.  that an intact C2 is not expressed by CLCuBuV. However, the limited number of clones analyzed here and the limitations of the RLM-RACE technique, namely that it cannot produce the sequences of full length transcripts, means that the results must remain tentative. It may be desirable to further analyse CLCuBuV transcription, using for example circularization reverse transcriptase PCR, to determine the full length sequences of viral transcripts and identify any possible low abundance transcripts. Moreover, northern blotting and quantitative RT-PCR would also be helpful to compare the expression of early and late genes at various stages of replication cycle.
The transcription of CLCuBuV and the recombinant CLCuMuB is equivalent to earlier begomoviruses/betasatellites that were transcription mapped. The recombinations/mutations that led to their appearance caused no detectable differences at the transcription level. Nevertheless, the study provides some avenues of investigation to follow-up in the efforts to determine the mechanism of resistance breaking in cotton by CLCuBuV and its betasatellite. These include the possible differences in the C4 protein, or expression thereof, and possible effects on REn expression, in addition to the hypothesis put forward by Amrao et al.  that the C2 protein may have been the avirulence determinant of the pre-resistance breaking virus species that was recognized by resistant cotton. It is also important to note that the CLCuBuV REn is chimeric, consisting of sequences derived from both CLCuMuV and CLCuKoV . These possibilities will be the subject of future investigation of the mechanism of resistance breaking in cotton.
FA was supported by the Higher Education Commission (HEC, Pakistan) under the ‘Indigenous 5000 Fellowship Scheme’. RWB was supported by the HEC under the ‘Foreign Faculty Hiring Scheme’. The work in the group of FV was funded by the HEC with a grant under the “International Research Support Initiative Programme” to FA.
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