«Abstract Kreuze, J.F. 2002. Molecular studies on the sweet potato virus disease and its two causal agents. Doctor’s dissertation. ISSN 1401-6249, ...»
Kreuze, J.F. 2002. Molecular studies on the sweet potato virus disease and its two
causal agents. Doctor’s dissertation.
ISSN 1401-6249, ISBN 91-576-6180-4
The studies presented in this thesis contribute to an increased understanding of the
molecular aspects, variability and interaction of the two most important viral
pathogens of sweet potato (Ipomoea batatas L): Sweet potato feathery mottle virus
(SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), which cause the severe sweet potato virus disease (SPVD) when co-infecting sweet potato plants. SPVD is the most important disease affecting sweet potato in Africa, and may be the most important virus disease of sweet potato globally.
The coat protein gene sequences of several African SPFMV isolates were determined and compared by phylogenetic analyses. Results showed that East African SPFMV isolates were genetically distinct. They could furthermore be divided into two serotypes which differed in their ability to systemically infect the sweet potato cultivar Tanzania.
The aetiology of SPVD was studied in sweet potato plants co-infected with SPFMV and SPCSV using nucleic acid hybridisation, bioassays, tissue printing and thin section immunohistochemistry. Resistance to SPFMV in East African sweet potato cultivars was found to be due to inhibition of virus replication rather than movement and resistance was suppressed by infection with SPCSV, resulting in a ca. 600-fold increase in titres of SPFMV. Furthermore, in SPVD affected plants SPFMV is detected outside of the phloem, whereas SPCSV is detected only inside the phloem, which suggests novel as yet unknown mechanisms how SPCSV synergises SPFMV.
The genomic sequence of SPCSV was determined. It was composed of two RNA molecules (9407 and 8223 nucleotides), representing the second largest (+)ssRNA genome of plant viruses. The genomic organization of SPCSV revealed novel features for the genus Crinivirus, such as i) the presence of a gene putatively encoding an ribonuclease III-like protein, ii) near-identical, 208 nucleotides long 3’-sequences on both viral RNAs, and iii) the placement of the SHP gene at a new position on the genome of SPCSV relative to other closteroviridae. Northern analyses showed the presence of several sub-genomic RNAs, of which the accumulation was temporally regulated in infected tissues. The 5’-ends of seven sub-genomic RNAs were determined using a PCR based method, which indicated that the sgRNAs were capped.
Keywords: Sweet potato feathery mottle virus, Sweet potato chlorotic stunt virus, Ipomoea batatas, genetic variation, Crinivirus, Potyvirus, viral synergism, genome structure, expression strategy, virus resistance.
Author’s address: Jan Kreuze, Department of Plant Biology, SLU, SE-750 07 Uppsala, Sweden. E-mail: Jan.Kreuze@vbiol.slu.se Contents Abbreviations, 8 Introduction, 9 Sweet potato, 10 Viruses of sweet potato, 11 Complex virus diseases of sweet potato, 13 Sweet potato feathery mottle virus, 14 The infection cycle of a potyvirus, 14 The Closteroviridae, 18 Sweet potato chlorotic stunt virus, 22 Genetic variability of RNA viruses, 23 Viral synergism, 24 Natural virus resistance in plants, 24 Gene-for-gene resistance, 25 Recessive resistance, 27 RNA silencing, 27 Genetically engineered virus resistance in plants, 30 Protein mediated PDR, 30 RNA mediated PDR, 31 Other approaches, 31 Virus resistance in sweet potato, 32 Transgenic resistance, 32 Aims of the study, 34 Results and discussion, 35 Genetic and biological variability of SPFMV in East Africa, 35 Studies on the synergistic interaction between SPFMV and SPCSV, 37 Characterization of SPCSV, 38 Genetic variability of SPCSV, 40 Current and future studies, 41 Attempts to identify the SPCSV genes involved in synergism, 41 Towards transgenic resistance to SPVD, 44 Conclusions, 47 References, 48 Acknowledgements, 63 Appendix Papers I-IV The present thesis is based on the following papers, which will be referred to by
their Roman numerals:
I. Kreuze, J.F., Karyeija, R.F., Gibson, R.W. & Valkonen J.P.T. 2000.
Comparisons of coat protein gene sequences show that East African isolates of Sweet potato feathery mottle virus form a genetically distinct group. Archives of Virology 145, 567-574 II. Karyeija, R.F., Kreuze, J.F., Gibson, R.W. & Valkonen, J.P.T. 2000. Two serotypes of Sweetpotato feathery mottle virus in Uganda and their interaction with resistant sweetpotato cultivars. Phytopathology 90, 1250-1255 III Karyeija, R.F., Kreuze, J.F., Gibson, R.W. & Valkonen J.P.T. 2000.
Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants. Virology 269, 26-36 IV Kreuze, J.F., Savenkov, E. I. & Valkonen, J.P.T. 2002. Complete genomic sequence and analyses of subgenomic RNAs of Sweet potato chlorotic stunt virus reveals several new features for the genus Crinivirus. Submitted
Introduction Viruses are sub-microscopic, obligate intracellular parasites that infect every class of living organisms known to date. Viruses themselves are not living organisms and they occupy a unique position in biology. Since they are not functionally active outside of their host cells, they lead, at most, only a kind of borrowed life (van Regenmortel et al., 2000). Hull (2002) defines a virus as follows: A virus is a set of one or more nucleic acid template molecules, normally encased in a protective coat or coats of protein or lipoprotein, that is able to organize its own replication only within suitable host cells. It can usually be horizontally transmitted between hosts. Within such cells, virus replication is (1) dependant on the host’s protein synthesizing machinery, (2) organized from pools of the required materials rather than by binary fission, (3) located at sites that are not separated from the host cell contents by a lipoprotein bilayer membrane, and (4) continually giving rise to variants through various kinds of changes in the viral nucleic acid.
Peter Medawar, awarded the Nobel Prize for Medicine and Physiology in 1960 had another definition for viruses: A piece of nucleic acid surrounded by bad news! Indeed, most viruses cause disease. By utilizing cellular substances and disrupting cellular processes, viruses cause the host metabolism to get upset, leading to development of abnormal compounds and conditions injurious to the functions and the life of the infected organism. Viral diseases such as influenza, polio, rabies, smallpox and AIDS have, and will continue to kill a countless number of people throughout the world. But viruses do not only cause human suffering directly. By infecting our livestock and crops, they can cause enormous economic losses and even hunger and starvation. Some virus diseases have destroyed entire plantings of certain crops in some areas, for example, geminiviruses in tomato, plum pox, hoja blanca of rice, Cacao swollen shoot, rice tungro, papaya ringspot, sugar beet yellows and citrus tristeza (Agrios, 1997; Bos, 1999). Because developing countries lack resources to control or limit damage caused by viruses, they often suffer most. Maize streak viruses cause severe yield losses yearly in Africa and since 1988 an epidemic of the African cassava mosaic virus (ACMV) has caused the complete collapse of cassava production in several districts of Uganda and western Kenya, leading to food shortages and famine (Otim-Nape et al., 2000). How many lives were lost due to the indirect effects of malnutrition or what kind of impact these viruses have had on a largely subsistent society with an already weak economy is unknown, but it is bound to be significant. The loss of the cassava crop due to ACMV has lead farmers to switch to other crops, such as sweet potato. Sweet potato, however, in its turn, is also affected by a severe viral disease.
Virus infected plants cannot be cured, and the only way to adequately protect the crops of subsistence farmers is by the use of resistant cultivars. We, as scientist, can contribute to reducing human hardship by developing and making available such resistant cultivars to those who are in need of them. This is not an easy task, viruses are continually changing, exploring new sequence space to adapt to the alterations in their hosts, and resistance-breaking strains appear. The development of durable resistance will be more likely if it is based on a thorough understanding of the pathogens involved, and the mechanisms by which they cause disease. The studies presented in this thesis contribute to an increased understanding of the molecular aspects, variability and interaction of the two most important viral pathogens of sweet potato: Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), which cause the severe sweet potato virus disease (SPVD) when co-infecting sweet potato plants.
Sweet potato Sweet potato (Ipomoea batatas L.) is a dicotyledonous, perennial plant, producing edible tuberous roots. It belongs to the family Convolvulaceae, the Morning Glory (Austin 1987). This family contains about 55 genera (Watson & Dallwitz, 2000).
The genus Ipomoea is thought to contain over 500 species with ploidy levels ranging from 2x to 6x (Ozias-Akins & Jarret, 1994). Sweet potato is the only Ipomoea species of economic importance as a food crop (Onwueme & Charles, 1994), and has both 4x and 6x forms (2n = 4x = 60 or 2n= 6x = 90). I. batatas probably originates from a cross between the ancestors of I. trifida (Huang & Sun, 2000; Jarret & Austin, 1994) and another wild Ipomoea sp., in Central or northern South America, at least 5000 years ago, and may be among “man’s” earliest domesticates. By the time of European contact, sweet potato was cultivated throughout the American tropics and had spread to the Easter Islands, Hawaiian and other Polynesian islands, as well as New Zealand. Sweet potato was introduced several different times into Europe, Africa and Asia during the late 15th and 16th centuries. In Africa, sweet potato was introduced to at least two places, West Africa and East Africa (Austin 1987).
Today, thousands of cultivars of sweet potato are grown throughout the tropics and subtropics (He, Prakash & Jarret, 1995). With an annual production of more than 133 million tons globally, sweet potato currently ranks as the seventh most important food crop on a fresh-weight basis in the world, and fifth in developing countries after rice, wheat, maize, and cassava (CIP,1999a). The production is concentrated in East Asia, the Caribbean, and tropical Africa, with the bulk of the crop (88%) being grown in China (Fig. 1; Hijmans, Huaccho & Zhang, 2001). In Africa the production is concentrated in the countries around the Lake Victoria.
Uganda is the biggest producer of sweet potato in Africa, and the third in the world. Sweet potato is processed into snacks, starch, liquor, flour and a variety of other industrial products. In addition to being used for human consumption, sweet potato is also widely used as an animal feed (CIP, 2000b). Because of the enormous genetic diversity of sweet potato (Zhang et al., 1998, 2000), and the accompanying diversity in phenotypic and morphological traits (Woolfe, 1992), the crop has great potential for further development to accommodate specific uses.
Sweet potato performs well in relatively poor soils, with few inputs, and has a short growing period. Among the major starch staple crops, it has the largest rates of production per unit area per unit time (Woolfe, 1992): in some areas up to three harvests per year can be achieved (Karyeija, Gibson & Valkonen, 1998a). Sweet potato tubers are rich in vitamin C and essential mineral salts. Due to the high beta-carotene content of yellow and orange-fleshed tubers, they are being promoted to alleviate vitamin A deficiency in East Africa (CIP, 1999b).
Despite the advantages that the cultivation of sweet potato offers, production tends to be concentrated in countries with low per capita incomes, and within those countries in regions where income levels are relatively low. Because of this, sweet potato has commonly been categorized as a “subsistence”, “food security”, or “famine relief” crop. Efforts to improve the agronomic qualities of sweet potato will therefore be of most benefit to developing countries, and particularly the poor sectors of the population within those countries. The International Potato Center (CIP), in Lima, Peru, has the international mandate for research on sweet potatoes in developing countries. Woolfe (1992) has reviewed the general agronomic principles of sweet potato production. The subsistence production of sweet potato in Africa has been reviewed by Karyeija, Gibson & Valkonen (1998a).
Fig. 1. Area cultivated with sweet potato over the period 1998-2000, each dot represents 1000 ha. (Hijmans, Huaccho & Zhang, 2001).
Viruses of sweet potato Although the sweet potato weevils (Cylas brunneus and C. puncticolis) are the most devastating pests of sweet potato worldwide (CIP, 2000a), diseases caused by viruses follow closely in importance wherever sweet potato is grown.
Worldwide at least 19 different viruses have been described in sweet potato, but only 11 of these have currently been recognized by the International Committee of Taxonomy of Viruses (ICTV; Table 1). This number, however, will most likely increase by additional surveys. Vegetative propagation, usually by taking cuttings from a previous crop (Onwueme & Charles, 1994; Karyeija, Gibson & Valkonen, 1998a), increases the risk of a build-up of viruses. The importance of virus diseases and their build-up in farmers’ planting material has been shown in China, where crops planted using pathogen tested sweet potato cultivars yielded 30-40% more, on average, than crops grown from farm-derived planting materials (Carey et al., 1999; Fugli et al., 1999).
Table 1. Viruses that have been reported in sweet potato crops