Accumulation and transmission of alphasatellite, betasatellite and tomato yellow leaf curl virus in susceptible and Ty-1-resistant tomato plants
Introduction
Viruses of the genus Begomovirus are circular single-stranded plant DNA viruses belonging to the family Geminiviridae, and are transmitted by the whitefly Bemisia tabaci (Zerbini et al., 2017). Their genome consists either of two components of about 2.6 kb (DNA-A and DNA-B components) or a single A-like component (Dry et al., 1993; Kheyr-Pour et al., 1991; Navot et al., 1991). Monopartite begomoviruses are often associated with alphasatellites or betasatellites, two types of circular ssDNA molecules of about 1.3 kb, detected mainly in Asia and Africa (Briddon and Stanley, 2006; Zhou, 2013). These complexes can cause important economic damage to crops.
The only sequence homology between begomovirus and betasatellite DNAs is within the conserved hairpin structure of begomovirus genomes (Briddon et al., 2001) that is required for rolling-circle replication (Heyraud-Nitschke et al., 1995; Laufs et al., 1995). Begomoviruses assist betasatellites for replication, and encapsidation (Dry et al., 1993; Saunders et al., 2000) with no strict specificity (Saunders et al., 2008). Betasatellite genome encodes the multifunctional protein βC1, which was reported to enhance symptom expression (Cui et al., 2004; Saunders et al., 2004), to suppress transcriptional gene silencing (TGS) (Yang et al., 2011b) and post transcriptional gene silencing (PTGS) (Amin et al., 2011; Cui et al., 2005), and to be involved in virus movement (Patil and Fauquet, 2010; Saeed et al., 2007). For some begomovirus species, e.g., Cotton leaf curl virus, Tomato yellow leaf curl China virus or Ageratum yellow vein virus, betasatellites are required for the expression of wild-type symptoms in their natural host plant (Cui et al., 2004; Mansoor et al., 1999). Betasatellites generally enhance virus accumulation (Jyothsna et al., 2013; Kon et al., 2009; Kumar et al., 2013; Saunders et al., 2000; Tiwari et al., 2010), but in some cases have no effect (Ranjan et al., 2014; Wu et al., 2011; Zhang et al., 2009). The accumulation and whitefly transmission of tomato leaf curl New Delhi virus (ToLCNDV) DNA was increased in the presence of the cotton leaf curl Multan betasatellite (CLCuMB) (Sivalingam and Varma, 2012).
The alphasatellite genome encodes a replication-associated protein (alpha-Rep) that resembles the Rep protein of nanoviruses (Mansoor et al., 1999; Saunders and Stanley, 1999). Alphasatellites are autonomous for their replication (Mansoor et al., 1999; Saunders and Stanley, 1999), but depend on a helper begomovirus for encapsidation, systemic infection and insect transmission. The effect of alphasatellites on helper virus is much less pronounced than that of betasatellites. Indeed, modulation of virus symptoms has been reported only rarely (Idris et al., 2011; Wu and Zhou, 2005) and the impact on virus accumulation is generally low or not detected (Briddon et al., 2004; Kumar et al., 2015, 2014). However, in a few instances, alphasatellites have been reported to induce a significant decrease in virus (Kon et al., 2009; Saunders et al., 2002; Wu and Zhou, 2005) and betasatellite accumulations (Idris et al., 2011; Kon et al., 2009; Wu and Zhou, 2005). Two alphasatellites, Gossypium darwinii symptomless alphasatellite and Gossypium mustelinium symptomless alphasatellite, have been shown to have strong gene silencing activities (Nawaz-ul-Rehman et al., 2010) and recently, alpha-Rep proteins of seven genetically different alphasatellite genomes were shown to restore the expression of a transcriptionally silenced GFP transgene in Nicotiana benthamiana (Abbas et al., 2017). These results suggest that alphasatellites are involved in overcoming host defense which could explain their frequent association with begomovirus/betasatellites complexes (Briddon et al., 2004; Xie et al., 2010).
Tomato yellow leaf curl virus is responsible for major damage in tomato crops worldwide (Moriones and Navas-Castillo, 2000). It was shown to originate in the Middle East (Lefeuvre et al., 2010), and seven strains are reported (Zerbini et al., 2017). The most widely distributed strains are the type strain of tomato yellow leaf curl virus (hereafter TYLCV-IL) and the Mild strain (TYLCV-Mld), which are predominant in the Mediterranean basin. The first report of natural association of TYLCV with a DNA satellite was from Oman where two tomato plants were found to be coinfected with TYLCV-OM (previously called ToLCOMV), TYLCV-Ir (previously called TYLCV-OM), tomato leaf curl betasatellite (ToLCB) and Ageratum yellow vein Singapore alphasatellite, and four plants with TYLCV-Ir and ToLCB (Idris et al., 2011; Khan et al., 2008). Other associations were detected in tomato in Jordan, Saudi Arabia and probably Egypt with isolates of Cotton leaf curl Gezira betasatellite (Abdel-Salam et al., 2017; Anfoka et al., 2014; Sohrab et al., 2017), and in Japan with Sida yellow vein China alphasatellite in a few samples of tomato (Shahid et al., 2014) and Cucurbita maxima (Shahid et al., 2015). Experimentally, TYLCV was shown to be a helper of any of the various betasatellites with which it was co-inoculated (Ito et al., 2009; Kon et al., 2009; Ueda et al., 2012; Zhang et al., 2009), and, most importantly, all of them increased its virulence. In view of these results, the introduction of betasatellites into western Mediterranean countries where satellites have never been reported is a potential threat to tomato cultivation.
The threat may be important if TYLCV/satellite associations are maintained in a sustainable way in the environment. As the sampling efforts in Middle Eastern countries are presently too limited to reveal if TYLCV-satellite associations are transient or not, it is proposed to address this question with experimentally infected plants, in which the accumulation level of viruses and satellites were monitored over time in comparison with that of TYLCV, and from which transmission efficiency of satellites was determined. Although it is known that alphasatellites and betasatellites can be assisted by various TYLCV strains in tomato plants and transmitted by B. tabaci (Ueda et al., 2012), with some impact on the accumulation of the helper virus, their accumulation level has not been compared so far to that of the helper virus during the infection course. Another factor that may determine the seriousness of the betasatellite threat is its potential capacity to overcome the popular Ty-1 resistance gene that is used in virtually all TYLCV-resistant tomato cultivars in the Mediterranean countries.
Experimental studies were conducted with TYLCV-Mld and TYLCV-IL as helper viruses, and three satellites from Burkina Faso: Cotton leaf curl Gezira betasatellite, and two alphasatellites, isolated from okra plants infected with Cotton leaf curl Gezira virus (Tiendrébéogo et al., 2010). It is noteworthy that Cotton leaf curl Gezira betasatellite (CLCuGB) has been reported previously in tomato plants infected with TYLCV (Anfoka et al., 2014). The two alphasatellite species are Cotton leaf curl Gezira alphasatellite and Okra leaf curl Burkina Faso alphasatellite. Although they were isolated from the same host species and country, their genomes exhibited 48% nucleotide divergence. The three satellites were inoculated with TYLCV-Mld in different combinations to susceptible tomato plants. Accumulation levels of virus and satellites were estimated by quantitative PCR (qPCR) in leaf samples collected between 11 and 150 days post inoculation (dpi), and compared to each other within and between treatments. The efficiency of transmission was estimated at 32 and 150 dpi for CLCuGB and at 32 dpi for OLCBFA. The capacity of TYLCV-Mld and TYLCV-IL to maintain the betasatellite was studied in a susceptible and a Ty-1 resistant cultivar by estimating their respective accumulations during infection.
Section snippets
Viruses and DNA satellites
TYLCV-Mld (TYLCV-Mld[RE:02] accession no. AJ865337) isolated from tomato plants (Solanum lycopersicum L.) in Réunion island (Delatte et al., 2005) was tested as helper virus with three DNA satellites isolated from okra in Burkina Faso (Tiendrébéogo et al., 2010): the betasatellite CLCuGB (CLCuGB-[BF:Kap:Ok1-2:08], FN554575), and two alphasatellites, cotton leaf curl Gezira alphasatellite (CLCuGA-[BF:Kap:Ok7:08], FN554580) and okra leaf curl Burkina Faso alphasatellite (OLCBFA-[BF:Pô:Ok1:08], FN554581
Impact of DNA satellites on symptoms and plant growth in TYLCV-Mld infected plants
Tomato plants were agroinoculated with TYLCV-Mld, either alone (TM), or together with the betasatellite CLCuGB (TM/B), the alphasatellites CLCuGA (TM/CA) or OLCBFA (TM/OA), or with a combination of CLCuGB and each alphasatellite (TM/B/CA, TM/B/OA) (Table 1, experiment 1). As agroinoculation was not 100% successful, some of the inoculated plants were not infected, or at least not infected with all the components in the mixed inoculations. Only plants which were detected positive for all
Discussion
The seriousness of the “satellite risk” to TYLCV infected tomato was assessed by testing experimentally two criteria associated with their maintenance: their accumulation level with respect to that of the helper virus, and their efficiency to be transmitted by their natural whitefly vector at an early and later stage of infection. To increase the relevance of the study, accumulation studies were performed with susceptible and Ty-1 resistant cultivars because virtually all TYLCV-resistant
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
Déborah Conflon is a PhD student from Montpellier SupAgro, France. She received a grant from CIRAD (http://www.cirad.fr/) and ANSES (https://www.anses.fr). The authors are grateful to Jean-Luc Macia and Sophie Le Blaye for their excellent technical assistance in growing and sampling the plants. Gautier Semences (Eyrargues, France) supported the project and supplied the tomato seeds of “Monalbo”, “Pristyla” and its nearly isogenic susceptible cultivar. All experiments were done in P3 containment
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Characterization of begomoviruses and DNA satellites associated with tomato
2022, Geminivirus: Detection, Diagnosis and ManagementFactors controlling the fate of tomato yellow leaf curl virus (TYLCV) in its vector, the whitefly vector Bemisia tabaci
2021, Plant Virus-Host Interaction: Molecular Approaches and Viral EvolutionTomato Yellow Leaf Curl Virus: Impact, Challenges, and Management
2020, Trends in Plant ScienceCitation Excerpt :Several β-satellites, like Cotton leaf curl Gezira betasatellite, Tobacco leaf curl Japan betasatellite, Honeysuckle yellow vein mosaic betasatellite, and Tomato leaf curl Philippines betasatellite, have been found to be associated with TYLCV [23,24]. An additional satellite DNA known as α-satellite, which is about 1.4 kb, has recently been found to be associated with TYLCV [17,25]. Like β-satellites, they are dependent on the helper virus for encapsulation, movement within plant, and transmission by vectors, but they can autonomously replicate within their hosts.
Geminivirus-Associated Betasatellites: Exploiting Chinks in the Antiviral Arsenal of Plants
2019, Trends in Plant ScienceCitation Excerpt :The Ty-1/Ty-3 genes in tomato were shown to code for a DFDGD-class RDR and to confer resistance to tomato yellow leaf curl virus (TYLCV) by increasing the level of cytosine methylation associated with the viral genome [24]. However, the Ty-1/Ty-3-derived anti-geminiviral resistance was compromised by mixed infection of TYLCV and cotton leaf curl Gezira betasatellite [25]. Considering the ability of βC1 to suppress DNA methylation-mediated TGS, it is noteworthy that there is a potential risk that co-infection with geminivirus–betasatellite disease complexes could result in a breakdown of the popular Ty-1/Ty-3 resistance.