Effects of the mutation of selected genes of Cotton leaf curl Kokhran virus on infectivity, symptoms and the maintenance of Cotton leaf curl Multan betasatellite

Virus Research 169 (2012) 107–116

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Effects of the mutation of selected genes of Cotton leaf curl Kokhran virus on infectivity, symptoms and the maintenance of Cotton leaf curl Multan betasatellite Zafar Iqbal a , M. Naeem Sattar b , Anders Kvarnheden b , Shahid Mansoor a , Rob W. Briddon a,∗ a b

Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Jhang Road, Faisalabad, Pakistan Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Linnean Centre of Plant Biology in Uppsala, Swedish University of Agricultural Sciences, Uppsala, Sweden

a r t i c l e

i n f o

Article history: Received 5 April 2012 Received in revised form 13 July 2012 Accepted 17 July 2012 Available online 31 July 2012 Keywords: Begomovirus Betasatellite Mutation Symptoms Movement Replication

a b s t r a c t Cotton leaf curl Kokhran virus (CLCuKoV) is a cotton-infecting monopartite begomovirus (family Geminiviridae). The effects of mutation of the coat protein (CP), V2, C2 and C4 genes of CLCuKoV on infectivity and symptoms in Nicotiana benthamiana were investigated. Each mutation introduced a premature stop codon which would lead to premature termination of translation of the gene. Mutation of the CP gene abolished infectivity. However, transient expression of the CLCuKoV CP gene under the control of the Cauliflower mosaic virus 35S promoter (35S-KoCP ), at the point of inoculation, led to a small number of plants in which viral DNA could be detected by PCR in tissues distal to the inoculation site. Mutations of the V2, C2 and C4 genes reduced infectivity. The V2 and C2 mutants did not induce symptoms, whereas the C4 mutation was associated with attenuated symptoms. Infections of plants with the C4 mutant were associated with viral DNA levels equivalent to the wild-type virus, whereas viral DNA levels for the V2 mutant were low, detectable by Southern blot hybridisation, and for the C2 mutant were detectable only by PCR. Significantly, transient expression of the CLCuKoV C2 gene at the point of inoculation, raised virus DNA levels in tissues distal to the inoculation site such that they could be detected by Southern hybridisation, although they remained at well below the levels seen for the wild-type virus, but reduced the infectivity of the virus. These findings are consistent with earlier mutation studies of monopartite begomoviruses and our present knowledge concerning the functions of the four genes suggesting that the CP is essential for long distance spread of the virus in plants, the C4 is involved in modulating symptoms, the C2 interferes with host defence and the V2 is involved in virus movement. The results also suggest that the V2, C2 and C4 may be pathogenicity determinants. Additionally the effects of the mutations of CLCuKoV genes on infections of the virus in the presence of its cognate betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB), were investigated. Mutation of the C4 gene had no effect on maintenance of the betasatellite, although the betasatellite enhanced symptoms. Inoculation of the C2 mutant with CLCuMuB raised the infectivity of the virus to near wild-type levels, although the numbers of plants in which the betasatellite was maintained was reduced, in comparison to wild-type virus infections with CLCuMuB, and viral DNA could not be detected by Southern hybridisation. Transient expression of the C2 gene at the point of inoculation raised virus DNA levels in tissues distal to the inoculation site but also reduced the infectivity of the virus and the numbers of plants in which the betasatellite was maintained. CLCuMuB restored the infectivity of the V2 mutant to wild-type levels but only in a small number of plants was the satellite maintained and infections were non-symptomatic. Although inoculation of the CP mutant with CLCuMuB did not restore infectivity, co-inoculation with 35S-KoCP increased the number of plants in which the virus could be detected, in comparison to plants inoculated with the mutant and 35S-KoCP , and also resulted in two plants (out of 15 inoculated) in which the betasatellite could be detected by PCR. This indicates that the V2, C2 and almost certainly the CP are important for the maintenance of betasatellites by monopartite begomoviruses. The significance of these findings is discussed. © 2012 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. Tel.: +92 41 2651475. E-mail addresses: [email protected] (Z. Iqbal), [email protected] (M.N. Sattar), [email protected] (A. Kvarnheden), [email protected] (S. Mansoor), [email protected] (R.W. Briddon). 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.07.016

Viruses of the family Geminiviridae have small, single-stranded (ss)DNA genomes that are encapsidated in characteristic twinned quasi-icosahedral particles and are ascribed to one of the four

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genera (Topocuvirus, Curtovirus, Mastrevirus or Begomovirus) based upon genome arrangement, insect vector and sequence relatedness (Brown et al., 2012). The genus Begomovirus encompasses the economically most destructive geminiviruses that are transmitted by the ubiquitous whitefly Bemisia tabaci. All begomoviruses originating from the New World (NW) typically have genomes consisting of two components, referred to as DNA A and DNA B, both of which are required for virus infectivity. In the Old World (OW), although a small number of bipartite begomoviruses are known, the majority have only a single genomic component, a homolog of the DNA A of the bipartite viruses. A small number of these are truly monopartite, their single component genomes inducing disease in plants in the field, such as Tomato leaf curl virus (ToLCV) in Australia (Dry et al., 1993). Most monopartite begomoviruses instead associate with ssDNA satellites known as betasatellites (earlier referred to as DNA ␤) and satellite-like components known as alphasatellites (earlier referred to as DNA 1; Briddon and Stanley, 2006). The genomes of monopartite (and DNA A components of bipartite) begomoviruses are typically ∼2800 nucleotides in length and transcribe genes bi-directionally from a non-coding intergenic region which contains promoter elements and the origin of virion-strand DNA replication. The virion-sense strand encodes the coat protein (CP; required for insect transmission and movement in plants) and V2 protein (believed to be involved in virus movement in plants; Rojas et al., 2001). The complementarysense strand genes encode the replication-associated protein (Rep; the only virus-encoded gene product required for viral DNA replication, which is a rolling circle replication initiator protein; Hanley-Bowdoin et al., 2004), the C2 protein (which for some begomoviruses up-regulates the late, virion-sense genes [and is then known as the transcriptional activator protein; TrAP], is a suppressor of post-transcriptional gene silencing [PTGS; Yang et al., 2007] and also overcomes virus induced hypersensitive cell death [Hussain et al., 2007; Mubin et al., 2010]), the replication enhancer protein (that is involved in establishing an environment conducive for virus replication; Settlage et al., 2005) and the C4 protein (the function of which remains unclear but for some viruses is a pathogenicity determinant and a suppressor of PTGS; Gopal et al., 2007; Saeed et al., 2008; Vanitharani et al., 2004). Betasatellites are small (∼1350 nucleotides in length) ssDNA satellites that occur exclusively in the OW and associate with monopartite begomoviruses (Briddon et al., 2008). Since they were first identified in 1999 (Saunders et al., 2000), more than 400 full-length betasatellite sequences have been deposited with the databases. Although considerable advances have been made in determining the contributions made by betasatellites to begomovirus–betasatellite complexes, little is known about the contributions made by the virus for maintenance of betasatellites. All betasatellite functions have been shown to be mediated by the single product they encode, ␤C1, which include suppression of PTGS and extending virus host range (reviewed by Amin et al., 2010). Betasatellites are not capable of autonomous replication and thus rely on the virus-encoded Rep. However, the precise interactions between Rep and the betasatellite DNA to initiate replication remain far from clear at this time (Saunders et al., 2008). Several studies have examined the effects of mutagenesis of genes on the infectivity and symptoms of monopartite begomoviruses. These studies concerned the monopartite begomoviruses Tomato yellow leaf curl virus (TYLCV) and ToLCV which are adapted to plants of the family Solanaceae and do not associate with betasatellites (Rigden et al., 1993, 1994; Wartig et al., 1997). The study described here examined for the first time a monopartite, Malvaceae-adapted, betasatellite-associated begomovirus – Cotton leaf curl Kokhran virus (CLCuKoV). In addition to assessing the effects of mutagenesis of genes on infectivity and symptoms, the effects of

the mutations on maintenance of the cognate betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB), was assessed. 2. Materials and methods 2.1. PCR-mediated mutagenesis, production of constructs for infectivity and transient gene expression Specific gene mutations were introduced into a CLCuKoV clone (acc. no. AJ496286) which has previously been shown to be infectious (Mansoor et al., 2003). Mutants were produced by designing back-to-back oligonucleotide primers (Supplementary Table 1), containing the desired sequence changes, and using these to PCRamplify the complete viral genome. The mutations of the C2 gene were introduced into the area of the gene which does not overlap either the C1 or C3 genes. Mutation of the C4 gene consisted of a change from C to G at position 2297 introducing a stop codon in the C4 but only a silent change (no change in amino acid sequence) for the overlapping C1 gene. Resulting full-length virus clones were sequenced in their entirety to ensure no additional sequence changes were introduced. A partial direct repeat construct for the CLCuKoV clone bearing a mutation in the V2 gene (KoV2 ) was produced by cloning an approx. 250 bp BamHI–NotI fragment in pGreen0029 (Hellens et al., 2000). The full length genome, released as a BamHI fragment, was then cloned into the unique BamHI site of the pGreen0029 construct containing the 250 bp fragment. A similar strategy was followed for clones harbouring mutations of the CP gene (KoCP ; ∼650 bp KpnI–NotI fragment), the C2 gene (KoC2 ; ∼1400 bp XhoI–BamHI) and the C4 gene (KoC4 ; ∼550 bp XhoI–BamHI fragment). The production of a construct for the Agrobacterium-mediated inoculation of CLCuMuB (acc. no. AJ298903; Briddon et al., 2001), in the binary vector pGreen0029, has been described previously (Saeed et al., 2005). Constructs for the expression of CLCuKoV genes under the control of the Cauliflower mosaic virus (CaMV) 35S promoter were produced by PCR-mediated amplification of the coding sequences using specific oligonucleotide primers (Supplementary Table 1). Restriction endonuclease recognition sites for SalI and BamHI were included in the forward and reverse primers, respectively, to allow directional cloning in the expression vector pJIT163 (Guerineau et al., 1992). Resulting pJIT163 expression cassettes were transferred into the binary vector pGreen0029 as KpnI–EcoRV fragments. PCR-mediated amplifications, restriction endonuclease digestion, and cloning were conducted by standard methods with enzymes obtained from Fermentas (Arlington, Canada). 2.2. Agrobacterium-mediated inoculation Binary vector constructs were electroporated into Agrobacterium tumefaciens strain GV3101. Agrobacterium inocula were prepared and inoculated to plants, by infiltration, as described previously (Hussain et al., 2005, 2007). Plants were kept in an insect-free glasshouse at 25 ◦ C with supplementary lighting to give a 16 h photoperiod or a growth chamber set for an 18 h photoperiod (200 ␮/m2 /s light intensity), at 25 ◦ C day/22 ◦ C night temperature and a relative humidity of 65%. Following inoculation plants were observed daily for the appearance of symptoms. At 25–30 days post-inoculation (dpi) the plants were photographed and leaf samples were harvested to isolate DNA for PCR and Southern blot analysis. 2.3. PCR-mediated diagnostics and Southern blot hybridisation Total genomic DNA was isolated from plants as described previously (Dellaporta et al., 1983). PCR-mediated diagnostics was

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Table 1 Infectivity of CLCuKoV and single gene mutants in N. benthamiana in the presence and absence of CLCuMuB. Inoculuma

Symptomsb

Infectivity PCR diagnostics (plants infected/plants inoculated)

Expt. II

Expt. I

Ko Ko, C␤ KoV2 KoV2 , C␤ KoV2 , 35S-V2Ko KoV2 , 35S-V2Ko , C␤ KoCP KoCP , C␤ KoCP , 35S-CPKo KoCP , 35S-CPKo , C␤ KoC2 KoC2 , C␤ KoC2 , 35S-C2Ko KoC2 , 35S-C2Ko , C␤ KoC4 KoC4 , C␤ KoC4 , 35S-C4Ko KoC4 , 35S-C4Ko , C␤ Mock

Latent period (days)

Southern blot analysisc

Expt. III

Ko

C␤

Ko

C␤

Ko

C␤

5/5 5/5 3/5 5/5 2/5 3/5 0/5 0/5 1/5 2/5 2/5 5/5 1/5 2/5 5/5 5/5 5/5 5/5 0/3

– 5/5 – 5/5 – 1/5 – 0/5 – 0/5 – 3/5 – 1/5 – 5/5 – 5/5 –

5/5 5/5 2/5 5/5 3/5 2/5 0/5 0/5 1/5 1/5 2/5 4/5 1/5 2/5 5/5 5/5 5/5 5/5 0/3

– 5/5 – 5/5 – 1/5 – 0/5 – 1/5 – 3/5 – 1/5 – 5/5 – 5/5 –

5/5 5/5 2/5 5/5 2/5 3/5 0/5 0/5 2/5 2/5 4/5 5/5 2/5 3/5 5/5 5/5 5/5 5/5 0/3

– 5/5 – 5/5 – 2/5 – 0/5 – 1/5 – 4/5 – 2/5 – 5/5 – 5/5 –

Ko

C␤

(+) (+) (+) (+) (−) (+) (−) (−) (−) (−) (−) (−) (+) (+) (+) (+) (+) (+) (−)

– (+) – (−) – (−) – (−) (−) (−) – (−) – (−) – (+) – (+) –

LC, VT LC, VT, ST NS NS NS NS NS NS NS NS NS NS NS NS LC LC, VT, ST LC LC, ST NS

12 10 – – – – – – – – – – – – 13–14 11–12 14 11–12 –

a Viruses, mutants and the betasatellite are denoted as Cotton leaf curl Kokhran virus (Ko), Cotton leaf curl Kokhran virus with the V2 gene mutated (KoV2 ), the CP gene mutated (KoCP ), the C2 gene mutated (KoC2 ), the C4 gene mutated (KoC4 ) and Cotton leaf curl Multan betasatellite (C␤). b Symptoms are denoted as leaf curling (LC), vein thickening (VT), stunting (ST) and no symptoms (NS). c The detection of CLCuKoV (Ko) and CLCuMuB (C␤) by Southern hybridisation is indicated as either positive (+) or negative (−).

performed by using CP primers (Supplementary Table 1) for detection of Ko and mutants thereof, and ␤C1 primers for the detection of CLCuMuB from isolated DNA (Supplementary Table 1). Total nucleic acid (10 ␮g) was resolved by electrophoresis in 1.5% agarose gels in 0.5× TAE buffer and transferred to nylon membranes (Hybond XL, Amersham). Viral DNA was detected by using the PCR-amplified, radioactively labelled CP gene fragment and the betasatellite was detected by using the ␤C1 gene fragment. DNA fragments were radioactively labelled with [␣-32P]dCTP by the random primer method using the “Rediprime II DNA Labeling System” (Amersham). Southern blot transfer and hybridisation were performed as described previously (Sambrook et al., 1989). Hybridisation signals were detected using a phosphorimager (Bio-Rad Personal FX Phosphorimager). 3. Results 3.1. Effects of mutation of the V2 gene of CLCuKoV on infectivity, symptoms and the ability to maintain CLCuMuB CLCuKoV (Ko) was highly infectious to Nicotiana benthamiana plants by Agrobacterium-mediated inoculation (Table 1) and, on the undersides of leaves developing subsequent to inoculation, induced downward leaf curling, crumpling and thickening of veins at 12 dpi (Fig. 1). Subsequently symptoms became progressively more severe with pronounced leaf curling, a reduction in leaf size and stunting of growth, in comparison to non-inoculated plants (Fig. 1). More severe symptoms, with the outer edges of leaves developing after inoculation being rolled upwards but with the leaves showing a downward cupping with swollen veins, were exhibited by all N. benthamiana plants when Ko was co-inoculated with CLCuMuB (C␤; Table 1). However, the presence of the betasatellite reduced the latent period (10 days), in comparison to plants inoculated with only Ko (12 days), and older plants showed a pronounced chlorosis. Southern blot analysis showed no significant difference in the amount of viral DNA accumulating in plants when the virus infection was with or without the betasatellite (Fig. 2A).

N. benthamiana plants inoculated with CLCuKoV harbouring a mutation of the V2 gene (KoV2 ) did not develop symptoms. However, by PCR, the presence of virus was detected in half of the plants inoculated (Table 1) and Southern blot analysis showed the presence of greatly reduced viral DNA levels in tissues developing subsequent to inoculation in comparison to plants infected with the wild-type virus (Fig. 2A). This indicates that the mutant virus was able to move from the site of inoculation, but at reduced efficiency (fewer plants infected) in comparison to the wild-type virus. Although co-inoculation of KoV2 with C␤ also did not lead to symptoms, PCR showed the presence of both virus and betasatellite in all inoculated plants. For these infections also the viral DNA levels were very low (Fig. 2A) and the betasatellite was not detected by Southern hybridisation (Fig. 3). Noticeably there was an absence of ssDNA virus forms in all KoV2 infections in which the virus could be detected by Southern hybridisation. Co-inoculation of KoV2 with a construct for the expression of the CLCuKoV V2 gene under the control of the 35S promoter (35S-V2Ko ) to N. benthamiana did not lead to symptomatic infection and viral DNA was detected in half of the inoculated plants by PCR but not by hybridisation (Table 1; Fig. 2A). Inoculation of plants with KoV2 , 35S-V2Ko and C␤ similarly did not lead to symptomatic plants (Fig. 1). Although the virus was detected in 8 out of 15 inoculated plants by PCR, Southern hybridisation detected very low viral DNA levels but was not able to detect the betasatellite DNA (Table 1; Figs. 2A and 3). However PCR mediated diagnostics revealed that the betasatellite was maintained in 4 out of 15 plants (Table 1), suggesting that the betasatellite DNA levels were below the detection threshold for detection by Southern blotting. 3.2. Effects of mutation of the CP gene of CLCuKoV N. benthamiana plants inoculated with the CP mutant of CLCuKoV (KoCP ) did not develop symptoms. Viral DNA could not be detected by either PCR or Southern blotting (Fig. 2B), indicating

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Fig. 1. Effects of the mutation of selected genes of CLCuKoV on the symptoms induced by the virus in N. benthamiana plants in the presence and absence of CLCuMuB. Plants were either not inoculated (healthy; A) or inoculated with CLCuKoV (B), CLCuKoV and CLCuMuB (C), CLCuKoVV2 (D), CLCuKoVV2 and CLCuMuB (E), CLCuKoVV2 and 35S-V2Ko (F), CLCuKoVCP (G), CLCuKoVCP and CLCuMuB (H), CLCuKoVCP and 35S-CPKo (I), CLCuKoVCP , CLCuMuB and 35S-CPKo (J), CLCuKoVC2 (K), CLCuKoVC2 and CLCuMuB (L), CLCuKoVC2 and 35S-C2Ko (M), CLCuKoVC2 , CLCuMuB and 35S-C2Ko (N), CLCuKoVC4 (O), CLCuKoVC4 and CLCuMuB (P), CLCuKoVC4 and 35S-C4Ko (Q), and CLCuKoVC4 , CLCuMuB and 35S-C4Ko (R). Photographs were taken at 25 dpi.

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Fig. 2. Southern blot detection of CLCuKoV in N. benthamiana plants. Blots were probed with a radioactively labelled fragment spanning the CP gene. On all four blots the DNA samples run on the gels were extracted from the leaves of plants that were mock-inoculated (lane M), not inoculated (H) or inoculated with CLCuKoV (1), CLCuKoV and CLCuMuB (2). For panel A samples were from plants inoculated with CLCuKoVV2 (3,4), CLCuKoVV2 and CLCuMuB (5,6), CLCuKoVV2 and 35S-V2Ko (7), CLCuKoVV2 , CLCuMuB and 35S-V2Ko (8). For panel B the samples were from plants inoculated with CLCuKoVCP (3), CLCuKoVCP and CLCuMuB (4), CLCuKoVCP and 35S-CPKo (5), CLCuKoVCP , CLCuMuB and 35S-CPKo (6). For panel C samples were from plants inoculated with CLCuKoVC2 (3 and 4), CLCuKoVC2 and CLCuMuB (5 and 6), CLCuKoVC2 and 35S-C2Ko (7), CLCuKoVC2 , CLCuMuB and 35S-C2Ko (8). For panel D samples were from plants inoculated with CLCuKoVC4 (3), CLCuKoVC4 and CLCuMuB (4), CLCuKoVC4 and 35S-C4Ko (5) or CLCuKoVC4 , CLCuMuB and 35S-C4Ko (6 and 7). Approximately equal amounts of total DNA extract (10 ␮g) were loaded in each case. The sample in lane P consisted of the monomeric CLCuKoV clone in pTZ57R/T (100 ng) as a hybridisation control. Viral DNA forms are indicated as single-stranded (ss), super-coiled (sc) and open-circular (oc). The additional virus-specific band lower on the blot and not labelled likely consists of defective forms of the virus.

that the mutation abolished the ability of the virus to infect N. benthamiana. Similarly, co-inoculation of N. benthamiana plants with KoCP and C␤ also did not lead to symptomatic infection (Table 1). Neither Southern blotting nor PCR-mediated diagnostics detected either viral or betasatellite DNA in leaves developing subsequent to inoculation (Figs. 2B and 3). The CLCuKoV CP gene was expressed under the control of the CaMV 35S promoter (35S-CPKo ) to investigate the possibility of complementing the mutation of the CP. Co-inoculation of KoCP with 35S-CPKo to N. benthamiana did not lead to symptomatic infection. However, a very low level of infection could be detected by

PCR (4 out of 15 inoculated plants) but not by Southern hybridisation (Table 1; Fig. 2B). Similarly, when betasatellite was added to this combination (KoCP , 35S-CPKo and C␤), inoculated plants failed to show symptoms (Fig. 1). However, slightly more plants were infected (5 out of 15 plants) in comparison to plants inoculated with KoCP , 35S-CPKo (Table 1). Although the DNAs could be detected by PCR, they were not detected by Southern hybridisation, suggesting that DNA levels were below the detection threshold. These findings indicate that CP supplied in inoculated tissues complemented the ability of the virus (and betasatellite) to spread systemically but did not restore the ability to induce symptoms.

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the threshold for detection by hybridisation (Fig. 3). All plants inoculated with 35S-C2Ko showed a pronounced chlorosis (Fig. 1C, panel M) at the site of inoculation, which progressed to cell death (necrosis). Necrosis was not evident for the inoculations without this construct. 3.4. Effects of the mutation of the C4 gene of CLCuKoV

Fig. 3. Southern blot detection of CLCuMuB in N. benthamiana plants. Blots were probed with a radioactively labelled fragment spanning the ␤C1 gene. The DNA extracts run on the gel were extracted from the leaves of a mock-inoculated plant (M), a non-inoculated plant (H) and plants inoculated with CLCuKoV and CLCuMuB (lane 1), CLCuKoVV2 and CLCuMuB (2), CLCuKoVV2 , CLCuMuB and 35S-V2Ko (3), CLCuKoVCP and CLCuMuB (4), CLCuKoVCP , CLCuMuB and 35SCPKo (5), CLCuKoVC2 and CLCuMuB (6), CLCuKoVC2 , CLCuMuB and 35S-C2Ko (7), CLCuKoVC4 and CLCuMuB (8), and CLCuKoVC4 , CLCuMuB and 35S-C4Ko (9). Approx. equal amounts of total DNA extract (10 ␮g) were loaded in each case. Betasatellite DNA forms are indicated as single-stranded (ss), super-coiled (sc) and open-circular (oc).

3.3. Effects of mutation of the C2 gene of CLCuKoV Mutation of the C2 gene of CLCuKoV (KoC2 ) abolished the ability of the virus to induce symptomatic infection of N. benthamiana (Fig. 1). However, PCR-mediated amplification showed the presence of viral DNA in 8 out of 15 plants inoculated (Table 1). The viral DNA levels in these plants were, however, below the detection threshold of Southern blotting (Fig. 2C). Co-inoculation of N. benthamiana plants with KoC2 and C␤ similarly did not lead to symptomatic infection and no viral or betasatellite DNA could be detected by Southern hybridisation (Figs. 2C and 3). Nevertheless, a high proportion of plants (14 plants out of 15 inoculated) were shown to contain viral DNA by PCR (Table 1). Of these, 10 plants were shown also to contain the betasatellite (Table 1). This indicates that the betasatellite was able to complement the C2 mutation by increasing the infectivity comparable to wild type virus with respect to infectivity (increasing the number of plants that ultimately have virus in tissues distal to the inoculation site) but not symptoms. A construct for the expression of the C2 gene of CLCuKoV under the control of the 35S promoter (35S-C2Ko ) was used to try to complement the mutation. Inoculation of N. benthamiana plants with KoC2 and 35S-C2Ko did not lead to symptomatic infection but did result in viral DNA levels that could be detected hybridisation (Fig. 2C). PCR-mediated diagnostics showed the presence of viral DNA in 4 plants (out of 15 inoculated; Table 1), less than seen with plants inoculated with only KoC2 . Similarly, inoculation of the C2 mutant with C␤ and 35S-C2Ko did not lead to symptomatic infection (Fig. 1) and fewer plants ultimately showed the presence of the virus and the betasatellite by PCR (Table 1). Although for these plants virus levels were raised to such an extent that they could be detected by Southern blotting, betasatellite levels remained below

All N. benthamiana plants inoculated with the C4 mutant of CLCuKoV (KoC4 ) ultimately showed symptoms of infection (Table 1). However, the onset of symptoms was delayed by 1–2 days in comparison to plants inoculated with the wild-type virus and plants did not show thickening of veins on the undersides of leaves developing subsequent to inoculation (Fig. 1), which is typical of the virus with an intact C4. As for inoculations with KoC4 , all N. benthamiana plants inoculated with KoC4 and C␤ ultimately exhibited symptoms of infection and the symptoms were qualitatively and quantitatively indistinguishable from those induced by Ko in the presence of C␤ (Table 1; Fig. 1). However, the latent period for appearance of symptoms was less (11–12 days) than for KoC4 infections (13–14 days) but greater than for Ko and C␤ infections (10 days). Southern blotting showed the presence of both components at levels equivalent to those for inoculation with the wild type virus (Figs. 2D and 3). Plants inoculated with KoC4 and a construct for the expression of the C4 gene (35S-C4Ko ) showed the first symptoms at 14 dpi and were indistinguishable from plants inoculated with only KoC4 . No effect could be observed on vein thickening although C4 was expressed from a heterologous vector. Inoculation of KoC4 and C␤ with 35S-C4Ko to N. benthamiana plants induced typical leaf curling, with vein swelling and stunting symptoms at 11–12 dpi. Southern blot analysis of both groups of plants showed that the levels of both viral and betasatellite DNA were equivalent to those detected in plants inoculated with the wild type virus (Figs. 2D and 3). 4. Discussion The study described here was stimulated by the recent characterisation of Cotton leaf curl Burewala virus (CLCuBuV), and its betasatellite, that are associated with resistance breaking CLCuD across Pakistan and north-western India (Amrao et al., 2010; Rajagopalan et al., 2012; Zaffalon et al., 2011). This virus has a naturally mutated C2 gene. Although the C2 gene is experimentally dispensable for infection (Wartig et al., 1997), its maintenance in all characterised begomoviruses, curtoviruses and the only known topocuvirus indicates that it is important for survival in the field. This raised the question as to what effects mutations of virusencoded genes might have on the maintenance of a betasatellite. Additionally, previous studies that have mutated begomovirus genes have used Solanaceae-adapted viruses which are not associated with betasatellites. No such studies have looked at the Malvaceae-adapted viruses or viruses naturally associated with betasatellites. Mutation of the CP gene of CLCuKoV abolished the infectivity of the virus and co-inoculation with CLCuMuB did not complement the mutation – no plants showed movement of either virus or betasatellite by either Southern blot hybridisation or PCR. The CP has previously been shown to be essential for the infectivity of all classes of monopartite geminiviruses (begomo-, curto- and mastreviruses) (Boulton et al., 1989; Briddon et al., 1989; Rigden et al., 1993). It is presumed that, as well as playing a part as a movement protein (a nuclear shuttle-moving viral DNA, and presumably also satellite DNA, in and out of the nucleus), the CP is important for protecting viral DNA during long distance movement in the phloem, either as virus particles (virions) or as nucleoprotein

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complexes (Briddon and Markham, 2001; Gafni and Epel, 2002). It is thus unclear whether it is the “nuclear shuttle”, the “virus long-distance spread”, both or some other (as yet unidentified) function of the CP that the betasatellite is unable to complement. It has been shown that a betasatellite can complement the functions of DNA B, turning the DNA A of bipartite begomovirus into a monopartite virus (Saeed, 2010; Saeed et al., 2007). This finding led to the suggestion that betasatellites (more specifically the ␤C1) have “movement” functions. However, in this case the CP is still present. For all bipartite begomoviruses which have been investigated, the DNA A can move systemically, in the absence of DNA B, following Agrobacterium-mediated inoculation to N. benthamiana (Briddon and Markham, 2001; Evans and Jeske, 1993; Klinkenberg and Stanley, 1990). However, such infections are non-symptomatic and associated with very low viral DNA levels, consisting of mostly ssDNA and some double-stranded DNA. Also, the independent spread of DNA A has been shown to require the CP (Briddon and Markham, 2001), suggesting that viral long distance movement in the phloem is either as virions or nucleoprotein complexes. It is interesting to note, in the experiments conducted here, that transient provision of CP at the point of inoculation leads to a small number of plants in which the virus is detected by PCR. This suggests that CP is required to either gain access to the phloem, leading to virus being detectable in young developing tissue (but possibly not replicating), and/or that the CP is required to re-establish infection in the young tissue (thus not able to spread out of initially infected cells). Additionally it shows that a continual supply of CP is required to maintain an infection. Nevertheless, the detection of two plants in which the betasatellite could be detected at the top of the plant, when CP is provided transiently, indicates that the viral CP is important in the maintenance of betasatellites by begomoviruses. This would be expected since these satellites are entirely dependent on the helper virus for movement in plants and the virus has an absolute requirement for the CP for infectivity. The V2 gene mutant of CLCuKoV retained the ability to systemically infect N. benthamiana, albeit at a reduced efficiency (in comparison to the wild-type virus). However, infections did not induce symptoms and were associated with very low viral DNA levels. This is consistent with earlier mutational analyses of monopartite begomoviruses (Rigden et al., 1993; Wartig et al., 1997) and suggests that the V2 protein is involved in virus movement. The expression of V2 from a Potato virus X (PVX) vector resulted in severe leaf curling and suggests a role for V2 in symptom induction (Mubin et al., 2010). Inoculation of CLCuMuB with the CLCuKoV V2 mutant restored the infectivity of the virus (all inoculated plants became infected) but did not lead to symptoms and again virus, as well as betasatellite, DNA levels were very low. This indicates that, at least in part, the betasatellite (and thus likely the ␤C1 protein – the only protein encoded by betasatellites), can complement the V2 mutation. The V2 and ␤C1 proteins have several features in common – both bind nucleic acids, both are suppressors of PTGS, are implicated in virus movement, interact with the CP and have similar cellular localisations (Amin et al., 2011a; Cui et al., 2005; Poornima Priyadarshini et al., 2011; Sharma et al., 2011). Nevertheless, it is clear that at least one function of V2 cannot be complemented by ␤C1 and that V2 plays an important part in the maintenance of the betasatellite. It is possible that, as has been shown for the p25 triple gene block protein of potexviruses (Bayne et al., 2005), V2 suppresses a host PTGS-based resistance to virus movement. It is also important to note that a previous study of a bipartite begomovirus has noted that mutation of the V2 affects CP expression, although the precise mechanism was not defined (Bull et al., 2007). This possibility was not investigated here, although the apparent absence of viral ssDNA in KoV2 infected plants may suggest that the V2 mutation is affecting CP expression – bipartite

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begomoviruses with the V2 mutated typically producing little CP (Bull et al., 2007). Expression of the V2 gene from the 35S promoter upon inoculation of the CLCuKoV V2 gene mutant apparently reduced the levels of viral DNA to below those detectable by Southern hybridisation. The reason for this is unclear. Possibly spatially or temporally inappropriate expression of V2 interferes with virus infection reducing the amount of virus spreading out of the inoculated tissues. Certainly with the low amount of viral DNA detected for KoV2 infections it would not take much to push the levels below the detection threshold of hybridisation. However, in plants that additionally included the betasatellite, although viral DNA could be detected, the levels were lower than in inoculations without 35SV2Ko , indicating that the interference from exogenously expressed V2 is consistent. All begomoviruses native to the NW so far characterised are bipartite and lack the V2 gene. This has led to the suggestion that the absence of the V2, the gene product of which is required for virus movement, may be the reason for the apparent absence of monopartite begomoviruses in the NW. Nawaz-ul-Rehman et al. (2009) showed that CLCuMuB could be maintained by the NW bipartite begomovirus Cabbage leaf curl virus (CbLCuV) in the presence of the DNA B but not its absence. This contrasts with the ability of CLCuMuB to complement the DNA B of the bipartite OW begomovirus Tomato leaf curl New Delhi virus (Saeed et al., 2007) and the ability of CLCuMuB to complement (at least for infectivity) the V2 mutation shown here. This may indicate that the differences between NW and OW begomoviruses are more than just the absence of a gene. Possibly the absence of the V2 gene in NW viruses has led the DNA A to become more reliant on DNA B functions, reflected in the inability of CLCuMuB to complement (functionally replace) the DNA B of CbLCuV. The difference may also be reflected in the distinct, and conserved amino acid differences of the CP of viruses from these two regions (Ha et al., 2006) which acts cooperatively with V2 in virus movement. Mutation of the C2 gene of CLCuKoV significantly reduced the infectivity of the virus, as well as leading to non-symptomatic infections with low viral DNA levels. This contrasts with the results of Wartig et al. (1997) where a C2 mutation of the monopartite begomovirus TYLCV had little effect on the infectivity, ameliorated symptoms and did not significantly affect viral DNA levels in N. benthamiana. In the presence of CLCuMuB the infectivity of CLCuKoVC2 was restored to near wild-type levels, indicating that the betasatellite can, for the most part, complement the mutation. However, significantly fewer plants inoculated with CLCuKoVC2 and CLCuMuB ultimately maintained the betasatellite, in comparison to inoculations with the wild-type virus, indicating that C2 plays a part in the maintenance of the satellite. The begomovirusencoded C2 protein is a multifunctional protein. It is a transcription factor required for the expression of late (virion-sense) genes for bipartite begomoviruses (Gopal et al., 2007; Sunter and Bisaro, 1991), modulates host gene expression including micro RNA genes (Amin et al., 2011b; Trinks et al., 2005), can be a pathogenicity factor (Amin et al., 2011b; van Wezel et al., 2001), a suppressor of PTGS (Amin et al., 2011a; van Wezel et al., 2003), may counter programmed cell death (Mubin et al., 2010), conditions a virus nonspecific enhanced-susceptibility phenotype in transgenic plants (Sunter et al., 2001), and also interacts with and inactivates SNF1related kinase (Hao et al., 2003) and adenosine kinase (Wang et al., 2003). The apparent interference in virus infection (fewer plants infected with virus and betasatellite) when the C2 mutant, with or without the betasatellite, was co-inoculated with a construct for the constitutive expression of C2 could be due to inappropriate spatial or temporal expression of this important protein. However, it is more likely that the cell-death induced by the over-expression of C2, a feature also of the Cotton leaf curl Multan virus (CLCuMuV;

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another monopartite begomovirus associated with CLCuD) C2 protein (Amin et al., 2011b), prevented virus/betasatellite moving out of inoculated tissues. However, in plants where the C2 mutant virus was able to spread systemically after inoculation with a construct for the constitutive expression of C2, more viral DNA accumulated (sufficient for detection by hybridisation). This is consistent with the suggestion (outlined above for V2) that begomovirus spread is constricted by a host PTGS-based resistance but also with the recent evidence showing that the C2 of BCTV plays a part in establishing a cellular environment for efficient virus replication (Caracuel et al., 2012). Both these mechanisms (suppression of PTGS and enhancement of replication) might lead to the delivery of more viral DNA into the phloem. The findings with the C2 mutation of CLCuKoV here highlight the unusual nature of CLCuBuV. Although we have shown that for CLCuKoV the C2 is not absolutely required for infectivity (in N. benthamiana) and the loss of this gene product can be partly complemented by the betasatellite, the virus is clearly disabled – not inducing symptoms and accumulating to low levels. This clearly indicates that CLCuBuV is unusual and worthy of further investigation to ascertain how this virus copes with the apparent loss of such an important gene. The C4 protein in monopartite begomoviruses and curtoviruses is an important symptom determinant. Infections of N. benthamiana with CLCuKoV with the C4 gene mutation exhibited attenuated symptoms, in comparison to the wild-type virus, although qualitatively the symptoms in each case were similar except a lack of vein swelling with the mutant. Although the infectivity of the virus was not affected, the latent period increased by one to two days. Mutation of the C4 genes of TYLCV and ToLCV produced very much the same result (Jupin et al., 1994; Rigden et al., 1994) and suggests that the C4 protein of CLCuKoV is not involved in viral replication but may play a minor role in virus spread and in modulating symptoms. The C4 protein of BCTV, when expressed constitutively in transgenic plants induced hyperplasia (Latham et al., 1997). The lack of vein swelling for the CLCuKoV C4 mutant possibly indicates that this protein similarly induces cell proliferation. As for CLCuKoV, mutation of the C4 gene of BCTV abolished the vein swelling phenotype (Stanley and Latham, 1992) whereas mutation of the C4 of Beet severe curly top virus prevented infection of plants (Teng et al., 2010). However, PVX-mediated expression of the C4 gene of CLCuMuV or TYLCV did not induce hyperplasia in N. benthamiana (Amin et al., 2011b). This may indicate that the C4 proteins differ in their function/interactions between viruses, which is consistent with the sequences of these proteins not being well conserved (Rojas et al., 2005). Inoculation of the C4 mutant with CLCuMuB led to plants exhibiting more severe symptoms with sunken veins on the upper leaf surface and swollen on the lower leaf surface; symptoms both qualitatively and quantitatively indistinguishable from a normal CLCuKoV/CLCuMuB infection. This ability of the betasatellite to “override” the C4 phenotype has been shown previously with CLCuMuB and ToLCV – a monopartite begomovirus which does not naturally associate with betasatellites (Saeed et al., 2008). This may suggest that the C4 protein of CLCuKoV (and ToLCV) and the ␤C1 of CLCuMuB are functionally redundant (both inducing hyperplasia), although the precise function of the C4 remains elusive. Recently the C4 proteins of four begomoviruses (including the monopartite CLCuMuV and TYLCV) have been shown to modulate miRNA levels (Amin et al., 2011b), suggesting that C4 may be involved in modulating host gene expression to create an environment favourable for virus proliferation. However, it is clear that C4 is not required for the maintenance of the betasatellite in N. benthamiana. The study described here was conducted in the highly permissive experimental host N. benthamiana, a feature of this species attributed to a mutated RNA-dependent RNA polymerase 1 which

is important in host PTGS-mediated defence (Goodin et al., 2008; Yang et al., 2004). Previous mutational studies of begomoviruses have indicated that requirements for infectivity can be more stringent in the natural host. For example, the mutation of three genes of TYLCV gave different results in tomato (a few plants infected for only one mutant) than in N. benthamiana (2 mutants infectious) indicating that there is a large degree of host specificity for interactions. All mutational studies of the genomes of monopartite begomoviruses conducted previously have involved Solanaceae-adapted species (TYLCV and ToLCV), none have involved Malvaceae-adapted viruses (such as CLCuKoV) and one might thus expect the requirement for virus encoded genes to be more stringent. Unfortunately, at this time, there is no infectivity system available for cotton (Gossypium hirsutum – the natural host of CLCuKoV and CLCuMuB) to do a similar study in the natural host. The results of this study with a monopartite, betasatelliteassociated begomovirus are consistent with earlier studies with monopartite viruses which suggest that the CP and V2 proteins have a role in virus movement, the C4 may be a pathogenicity determinant responsible for (some of the) symptoms induced by the virus and the C2 protein is essential for inducing symptoms and thus also a pathogenicity determinant. Also the results indicate for the first time that, in addition to the Rep, the V2 and C2 proteins play a part in the maintenance in plants of betasatellites by monopartite begomoviruses. It is likely also that the CP is essential for betasatellite maintenance, although this could not be demonstrated directly due to this gene product being indispensable for the infectivity of the virus. In contrast, the C4 protein was shown not to be important for maintenance of the betasatellite, at least in N. benthamiana. This may indicate that, contrary to earlier evidence for TYLCV (Rojas et al., 2001) and ToLCV (Saeed et al., 2007), the C4 protein is not involved in virus movement or that C4 of CLCuKoV is distinct from that of TYLCV. The conclusions from the study presented here is that virusencoded proteins required for movement (CP and V2) are important in maintenance (movement) of a betasatellite in plants by a monopartite begomovirus. However, the finding that two of the three proteins implicated in betasatellite maintenance (V2 and C2), one of which is not implicated in virus movement, also have suppressor of PTGS activity is consistent with the hypothesis, first put forward by Amin et al. (2010), that begomovirus movement is countered by a host resistance based on gene silencing. This hypothesis was put forward following the demonstration that the ␤C1 protein encoded by betasatellites has both movement and suppressor of silencing activities (Saeed et al., 2007). The finding that C4 of CLCuKoV is not involved in maintenance of the betasatellite may suggest that it does not have suppressor activity, although the C4 protein of the related CLCuMuV does (Amin et al., 2011a), or that any suppressor activity it has does not counter the possible silencing-based resistance to virus movement. The validity of the “gene silencing-based host resistance to begomovirus movement” hypothesis may possibly be investigated using heterologous suppressor proteins – a study that has been initiated. The implications of this are that the interactions between betasatellites and their helper begomoviruses are more complex than previously suggested – thus not just trans-replication, for amplification of the DNA, and trans-encapsidation, for spread within and between plants. There are clearly multiple levels at which adaptation of a betasatellite to a helper begomovirus must occur for the interaction to be successful. Undoubtedly the host will also figure in these interactions and this requires further study. Having investigated in more detail the interactions between a betasatellite and its helper begomovirus, it will be interesting to see whether mutations of virus-encoded genes have the same effects on the second class of begomovirus associated satellites, the alphasatellites, as well as the ToLCV-sat-like molecules which,

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it is now becoming clear, are both more wide-spread and more diverse than initial results suggested (Dry et al., 1997; Fiallo-Olivé et al., 2012; Idris et al., 2011; Nawaz-ul-Rehman et al., 2010). The alphasatellites differ from betasatellites in encoding their own Rep, thus being capable of autonomous replication (Mansoor et al., 1999), but both types of molecules do not apparently have a strong selection mechanism for their maintenance (such as that provided by ␤C1 for betasatellites, at least in some hosts). These issues will be the focus of future studies. Acknowledgements ZI was supported by a PhD fellowship from the Higher Education Commission (HEC), Government of Pakistan. MNS was supported by the HEC/Swedish Institute under the “Overseas Scholarship Scheme for PhD in selected fields”. RWB is supported by the HEC under the “Foreign Faculty Hiring Program”. The work in Sweden was supported with a grant to ZI under the “International Research Support Initiative Program” (HEC) and a grant to AK from Magn. Bergvall’s Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.virusres.2012.07.016. References Amin, I., Hussain, K., Akbergenov, R., Yadav, J.S., Qazi, J., Mansoor, S., Hohn, T., Fauquet, C.M., Briddon, R.W., 2011a. Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus–betasatellite complex. Molecular Plant–Microbe Interactions 24, 973–983. Amin, I., Ilyas, M., Mansoor, S., Briddon, R.W., Saeed, M., 2010. Role of DNA satellites in geminiviral disease complexes. In: Sharma, P., Gaur, R.K., Ikegami, M. (Eds.), Emerging Geminiviral Diseases and their Management. Nova Science Publishers Inc., New York, pp. 209–234. Amin, I., Patil, B.L., Briddon, R.W., Mansoor, S., Fauquet, C.M., 2011b. Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs. Virology Journal 8, 238. Amrao, L., Amin, I., Shahid, S., Briddon, R.W., Mansoor, S., 2010. Cotton leaf curl disease in resistant cotton is associated with a single begomovirus that lacks an intact transcriptional activator protein. Virus Research 152, 153–163. Bayne, E.H., Rakitina, D.V., Morozov, S.Y., Baulcombe, D.C., 2005. Cell-to-cell movement of potato potexvirus X is dependent on suppression of RNA silencing. Plant Journal 44, 471–482. Boulton, M.I., Steinkellner, H., Donson, J., Markham, P.G., King, D.I., Davies, J.W., 1989. Mutational analysis of the virion-sense genes of maize streak virus. Journal of General Virology 70, 2309–2323. Briddon, R.W., Brown, J.K., Moriones, E., Stanley, J., Zerbini, M., Zhou, X., Fauquet, C.M., 2008. Recommendations for the classification and nomenclature of the DNA-ˇ satellites of begomoviruses. Archives of Virology 153, 763–781. Briddon, R.W., Mansoor, S., Bedford, I.D., Pinner, M.S., Saunders, K., Stanley, J., Zafar, Y., Malik, K.A., Markham, P.G., 2001. Identification of DNA components required for induction of cotton leaf curl disease. Virology 285, 234–243. Briddon, R.W., Markham, P.G., 2001. Complementation of bipartite begomovirus movement functions by topocuviruses and curtoviruses. Archives of Virology 146, 1811–1819. Briddon, R.W., Stanley, J., 2006. Sub-viral agents associated with plant single-stranded DNA viruses. Virology 344, 198–210. Briddon, R.W., Watts, J., Markham, P.G., Stanley, J., 1989. The coat protein of beet curly top virus is essential for infectivity. Virology 172, 628–633. Brown, J.K., Fauquet, C.M., Briddon, R.W., Zerbini, M., Moriones, E., Navas-Castillo, J., 2012. Geminiviridae. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy—Ninth Report of the International Committee on Taxonomy of Viruses. Associated Press/Elsevier Inc., London/Waltham/San Diego, pp. 351–373. Bull, S.E., Briddon, R.W., Sserubombwe, W.S., Ngugi, K., Markham, P.G., Stanley, J., 2007. Infectivity, pseudorecombination and mutagenesis of Kenyan cassava mosaic begomoviruses. Journal of General Virology 88, 1624–1633. Caracuel, Z., Lozano-Durán, R., Huguet, S., Arroyo-Mateos, M., Rodríguez-Negrete, E.A., Bejarano, E.R., 2012. C2 from Beet curly top virus promotes a cell environment suitable for efficient replication of geminiviruses, providing a novel mechanism of viral synergism. New Phytologist 19, 846–858.

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