Chapter 1 Introduction

Transcription

1 Chapter 1 Introduction Watermelon bud necrosis virus (WBNV), a member of the genus Tospovirus causes serious diseases in watermelon (Citrullus lanatus) in India. WBNV was first reported during 1990s in southern India (Singh and Krishnareddy, 1996) in watermelon growing areas, Karnataka, Andhra Pradesh and Maharashtra with an estimated yield loss of % (Krishnareddy and Singh, 1993). The affected plant produces a range of symptoms characterized by mottling and yellowing of leaves, stunting of vines and dieback of buds and shoots. The virus was initially identified as a member under the genus Tospovirus, family Bunyaviridae based on virion morphology, serology, host range and transmission by Thrips flavus (Singh and Krishnareddy, 1996). Subsequently, the virus was recognized as WBNV based on the characteristics of nucleocapsid (N) protein gene sequence, which shared a closer relationship with Watermelon silver mottle virus (WSMoV) and Groundnut bud necrosis virus (GBNV) (Jain et al., 1998). Tospoviruses have quasi-spherical enveloped virion of nm diameter and a tripartite ssrna genome designated as large (L), medium (M) and small (S) RNAs (Elliot, 1990). The L-RNA encodes RNA-dependent RNA-polymerase in viral complementary strand (Den Haan et al., 1991). The M-RNA encodes a movement protein (NSm) in viral sense and a precursor of the Gn and Gc glycoproteins in complementary sense (Kormlink et al., 1992). The S-RNA encodes the non-structural protein (NSs) in viral sense and nucleocapsid (N) protein in viral complementary sense (De Han et al., 1990). In India, tospoviruses are emerging constrains in fruits, pulses, oilseeds and vegetables. There are 19 species of the virus in the world. Among them, five distinct tospoviruses are reported from India, e.g., Capsicum chlorosis virus (CaCV) (Krishnareddy et al., 2008), GBNV (Reddy et al., 1992; Satyanarayana et al., 1996), Peanut yellow spot virus (PYSV) (Satyanarayana et al., 1998), Iris yellow spot virus (IYSV) (Ravi et al., 2006) and WBNV (Jain et al., 1998). Only GBNV and WBNV are most prevalent in India affecting leguminous, solanaceous and cucurbitaceous crops (Jain et al., 2007). GBNV is well characterized and

2 recognized as a distinct species under the genus Tospovirus whereas WBNV is a tentative member of Tospovirus. Watermelon (Citrullus lanatus (Thunb.) Matsun. & Nakai.) is a major cucurbit crop that accounts for 6.8 % of the world area devoted to vegetable production in A rough estimate of annual world value of watermelon exceeds US$14 billion (FASTAT, 2011). Watermelon is grown in more than 96 countries worldwide. China is the world leader in watermelon production with 63 % (63 million tones) of the total production in 2008 (FAOSTAT, 2011).Watermelon has the highest lycopene content among fresh fruits and vegetables. Lycopene in the human diet is associated with prevention of heart attacks and certain cancers. Watermelon rind contains an important natural compound called citrulline which is found in high concentration in the liver, and is involved with athletic ability and functioning of the immune system (Perkins-Veazie et al., 2001). Diseases caused by viruses are a major limiting factor in commercial watermelon production worldwide. Around the world, over 10 viruses are known to affect watermelon production (Provvidenti, 1986). In India, cultivation of watermelon is affected by many viral diseases: Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), Papaya ring spot virus W stain (PRSV-W) and Watermelon mosaic virus (WMV) (Bhargava et al.,1975; Vani and Varma, 1993) and WBNV (Singh and Krishnareddy, 1996). Virus diseases are destructive to the watermelon crop and are difficult to control. Chemical control of the insect vectors is not usually efficient for control of the disease. Natural resistance to several important viruses is not known or breeding programs have failed to produce cultivars with effective resistance in a reasonable period of time. The cultivars have been developed by traditional breeding to have resistance to watermelon fruit blotch (Rane and Latin, 1992), WMV (Gillaspie and Wright, 1993) and Zucchini yellows mosaic virus (Boyhan et al., 1992). Introduction of one or more genes (transgene) from one to another plants, which in many cases is not possible by conventional breeding methods because of sexual incompatibility, but can be achieved by plant transformation methods, based on DNA recombinant technology to produce the transformed/ transgenic plant. Such recombinant techniques have proved very useful in several ways: they offer the possibility of genetic diversity, save time by avoiding cross breeding, allow specific improvements in crops while 2

3 preserving the desirable characteristics of an inbred line, and create novel types of resistance genes that do not occur in nature. With the development of plant transformation and regeneration techniques, a number of plants have been transformed by introduction of foreign genes with the intention of getting resistance to viruses. Transformation of plants with fragments of viral genomes has been found to give rise to the lines of plants that are resistant to the virus from which the sequence was derived. Hamilton (1980) first raised the possibility of imparting resistance, called pathogen-derived resistance (PDR), to plants by transforming them with the genes from a pathogen. Sanford and Johnston (1985) postulated a generalized concept about PDR. Virus resistant cultivars are now being developed by introducing pathogen (virus)- derived genes into the existing elite commercial inbred hybrids by recombinant technology to produce the transgenic plants, which retain all the other characteristics of the parent. The first transgenic plant possessing virus gene-derived, genetically-engineered resistance was the tobacco plant expressing capsid protein (CP) gene of Tobacco mosaic virus (TMV) (Powell et al., 1986). This opened new horizons in the field of genetic engineering towards development of transgenic plants resistant to virus infections. Since then many other studies have reported that CP and other viral genes from other plant RNA viruses successfully protected the transgenic plant against respective virus infection. Pathogen derived resistance is broadly divisible into two types, viral protein mediated and RNA-mediated resistance (Baulcombe, 1996; Beachy, 1997; Ratcliff et al.,1997). The viral protein may or may not accumulate during viral protein mediated resistance, which is generally broad-based, protects the transgenic plants against several viruses, and is more commonly operative in transgenic plants. The second type, the RNA mediated resistance, appearing analogous to the gene silencing process in which expressions of an endogenous gene or a transgene is inhibited by the insertions of a homologous sequence or a non-translatable sequence or an intact gene in a sense orientation into the genome of a plant or transgenic plant. In the recent years, the application of pathogen-derived resistance (PDR) has emerged as an alternative to the conventional practices to control plant viruses. The transgenic plants 3

4 harbouring translatable version of N gene of tospoviruses show resistance against tospoviruses (Gielen et al., 1991, Pang et al., 1994, Prins et al., 1995), exhibit broad spectrum resistance (Pang et al., 1994), thought the level of protection moderate or low (Pang et al., 1994). On the other hand, non-translatable version of N gene (Haan et al., 1992; Pang, 1996; Prins, 1996) and NSm gene (Prins et al., 1997) can confer RNA mediated resistance against tospoviruses by triggering post-trascriptional gene silencing (PTGS) effective only against the closely related stains (De Hann 1992; Pang 1993). To improve watermelon resistance to environmental biotic and abiotic stresses and nutritional quality, genetic engineering approach is the most effective technology. The use of genetic transformation for production of transgenic plants and somaclonal variation to produce polyploidy plants appear to be the most common biotechnological approaches for cultivar improvement (Compton et al., 2004). Polyploid watermelons were found to be resistant to watermelon fruit blotch and nematodes (Garret et al., 1995) and triploid watermelons can improve flesh darkness, flavor and toughness of fruit skin (Raza et al., 2003). There are many efficient methods for regenerating watermelon (Compton et al., 1999; Chaturvedi and Bhatnagar, 2001; Sultana et al., 2004; Ibrahim et al., 2009). Stable genetic transformation of Citrullus spp. by Agrobacterium tumefaciens has been reported earlier (Ellul et al., 2003;Yalcin -Mendi et al., 2003; Akashi et al., 2005; Choi, 2008; Ibrahim et al., Recently, Park et al. (2005) have described the successful transformation of a watermelon rootstock (gongdae) by Agrobacterium tumefaciens-mediated transformation with the CGMMV-CP gene for developing virus resistance watermelon plant. This is the first report on the production of transgenic watermelon plants with virus resistance. There have been few reports of transgenic plants showing resistance to tospovirus (TSWV) in other parts the world using NP gene in tobacco (Gielen et al., 1991; MacKenzie and Ellis 1992; Pang et al., 1992), tomato plants (Ultzen et al., 1995) and peanut (Yang et al., 2004) and partial N gene in Nicotiana benthamiana ( Jan et al., 2000), while only one report from India is showing transgenic resistance in tobacco using N gene of PBNV (Venkatesan et al., 2009). As information on molecular characterization of WBNV and development of transgenic plants for resistance to WBNV are not available, the present study on molecular 4

5 characterization of an isolate of WBNV from northern India and development of transgenic watermelon plants using NP gene were undertaken with the following objectives: Objectives: 1. Molecular cloning and sequencing of M-segment of WBNV genome. 2. Analysis of phylogenetic relationships based on M-RNA sequence. 3. Development of transgenic construct based on sequence from M-RNA segment. 4. Transformation of watermelon with the transgene construct and analysis for its expression. 5