Towards durable resistance to apple scab using cisgenes Sameer Gururaj Joshi Thesis committee Thesis supervisor Prof. Dr. Ir. E. Jacobsen Professor of Plant Breeding (Genetical Variation and Reproduction), Wageningen University Thesis co-supervisors Dr. F.A. Krens Senior Scientist/Research group leader Plant Research International, Wageningen University Dr. Ir. H.J. Schouten Senior Scientist Plant Research International, Wageningen University Other members Prof. Dr. Ir. P.C. Struik, Wageningen University Prof. Dr. Ir. R. Rabbinge, Wageningen University Prof. Dr. C. Mariani, Radboud University Nijmegen Prof. Dr. C. Gessler, Swiss Federal Institute of Technology, Zurich This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences Towards durable resistance to apple scab using cisgenes Sameer Gururaj Joshi Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M. J. Kropff in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Thursday 27 May 2010 at 11 a.m. in the Aula Sameer Gururaj Joshi Towards durable resistance to apple scab using cisgenes, 137 pages Ph.D. thesis, Wageningen University, Wageningen, NL (2010) With references, with summaries in English and Dutch ISBN: 978-90-8585-661-0 Table of Contents Chapter 1 General Introduction 1 Chapter 2 Approaches for development of cisgenic apples 9 Chapter 3 Isolation and development of HcrVf transformants 25 Chapter 4 Resistance evaluation and expression analysis of HcrVf transformants 47 Chapter 5 Resistance spectrum of HcrVf genes to Venturia inaequalis 61 Chapter 6 Isolation and mapping of the novel apple scab resistance gene Vd3 77 Chapter 7 General Discussion 93 References 109 Summary in English 123 Summary in Dutch 127 Acknowledgements 131 Curriculum Vitae 135 List of publications 137 Educational statement Dedicated to my beloved parents Chapter 1 General Introduction General Introduction Apple crop Apple (Malus x domestica) is one of the important fruit crops of the world. The cultivated apple (Malus x domestica) belongs to the family Rosaceae, the sub-family Maloideae (Haris et al. 2002). It is the fourth most widely produced fruit crop in the world after bananas, oranges and grapes with a production of 66 million tonnes cultivated on 5 million ha (FAO, 2007). China is the largest producer (43%) with USA being the second largest producer (7%) of apple in the world (FAO, 2007). Apple has many health promoting compounds. It has very strong anti-oxidant activity, inhibits cancer cell proliferation, decreases lipid oxidation, and lowers cholesterol level. Apples contain a variety of phytochemicals, including quercetin, catechin, phloridzin, and chlorogenic acid, all of which are strong anti-oxidants (Boyer and Liu 2004). Apple production has suffered many drawbacks such as apple scab as fungal disease and fire blight as bacterial disease (Gessler and Patocchi 2007) resulting in huge loss in yield. Apple juvenility is the phase from seed germination untill the first flowering (Jonkers 1971). This period usually lasts four to six years (Webster 2005). This is a serious problem in conventional apple breeding. This can be overcome by physiological or transgenic approaches. The physiological method includes induction of early flowering by putting young seedlings in a climate chamber with high light intensity, high CO2 concentration and optimal temperature for shoot growth leading to many internodes. This led to flowering of one-year-old trees (Volz et al. 2007). The transgenic approach includes expression of BpMADS4 gene in apple, which led flowering in invitro shoots thirteen weeks after transformation (Flachowsky et al. 2009). Self incompatibility is another important problem which restricts fertilization and fruit setting in many tree fruit crops. Self incompatibility is governed by special site S-locus. This S-locus is a gene-complex with highly polymorphic which exists in many alleles. Apple trees display gametophytic self-incompatibility (GSI) (Broothaerts 2003). In GSI, pollen can germinate and penetrate the stigma, but a pollen tube cannot grow completely through the style, if its S-allele is equal to one of the S-alleles present in the style cells (Wertheim and Schmidt 2005). The two S-alleles in the style cells are of equal value (codominant), and thus both involved in affecting pollen-tube growth. They code for the production of enzymes called S-RNases (S for stylar and RNases for ribonucleases) that occur in the intracellular matrix of the transmitting tissue. In apple S-proteins are localized in the intercellular space of the transmitting tissue both in stigma and style (Certal et al. 1999). Self incompatibility can 1 Chapter 1 be over come by developing self fertile apples by silencing S-RNase gene (Broothaerts et al. 2004). Apple scab Apple scab is a fungal disease caused by Venturia inaequalis (Cke) Wint. In general this fungus takes nutrients through the active and the passive mechanisms. The active mechanism involves movement against concentration gradient which requires the direct input of substantial amounts of metabolic energy, ATP or proton gradients, to drive transportation. The passive mechanism is through diffusion, which does not require carriers. Such nutrients are usually soluble in lipids and can enter hydrophobic membranes, e.g. glycerol and urea (Waites et al. 2001). The pathogen V. inaequalis has two distinct phases, a obligate Biotrophic phase from the first ascosporic infection until leaf death or fruit maturity in which it growths subcuticularly on the host and a saphrophytic phase in old leaves and mature fruits where it can invade all the tissue. V. inaequalis obtains nutrients through an active mechanism rather than through a passive one (Nicholson and Rahe 2004). The crop loss due to apple scab may be more than 70% of total fruit value (Agrios 2005). Pathogenesis V. inaequalis has a hemibiotrophic life cycle characterized by a saprophytic phase in autumn-winter and a parasitic phase in spring-summer. The fungus passes the winter in the old fallen leaves as immature pseudothecia (fruiting body). Each pseudothecium contains 4 genetically different ascospore pairs derived from one meiosis (= 4) followed by one mitosis (4 x 2) resulting in eight ascospores per pseudothecium. Ascospores constitute the primary source of inoculum. Inoculation is brought about through the agency of the wind. For infection to occur, ascospores and conidia have to land on a young leaf or fruits of a compatible cultivar and need a water film to germinate. The ascospores are discharged into the air from the old leaves on the ground. Spore discharge is conditioned by (1) the maturity of the ascospores themselves. (2) The occurrence of rain-periods when the ascospores are mature. (3) Ascospores are also discharged by light. It has been estimated that 45 minutes of wet period at optimum time is enough to discharge eight billion ascospores from the fallen old leaves under a large apple tree. These ascospores require several hours of rainy conditions for germination. Within a few days, the pathogen establishes a food relation with the host (Hesler 1917). 2 General Introduction Spore germination: The conidia of V. inaequalis germinate readily in water. The conidia have very high specific gravity and on immersion in a drop of water they sink to the bottom of it. At this time conidia are attached to the surface on which it is resting. In a hanging drop, the germ tube continues to grow out as a long thin colorless hypha, but on the leaf desiccation, germ tube comes in contact with the cuticle and proceeds with the formation of “appressorium” (Wiltshire 1915). This penetrates the cuticle. Mycelium grows radially between the cuticle and epidermal cells from the point of invasion, partially dissolving the cuticle. The mycelium of the pathogen on the leaves and fruits, transforms into a stroma, a spore-bearing cushion (Hesler 1917). Penetration: Penetration of the cuticle and further growth of the fungus must be on young leaves and fruits. When the appressorium is firmly established growth of the hypha into the cuticle takes places, beginning as a small bulge from the centre of the attached disc. The hypha usually grows directly into the cuticle at right angles to the surface of the leaf. It is often enlarged as it enters to attach further. The hypha begins to grow between the epidermal wall and cuticle, forming a plate-like mycelium. Conidiogenesis: The subcuticular mycelium having been formed in the leaf, the uppermost cells of the stroma starts to enlarge, and push out the cuticle until they burst it open and form the conidiophores from which conidia begin at one end to be cut off (Wiltshire 1915). The conidia mature and will be dispersed to other leaves and fruits, and serve as secondary source of infection. From these secondary infection spots, caused by mycelial growth from the conidia, stromata arise on which a second crop of conidia are produced. This process repeats itself throughout the growing season. As the leaves fall, the mycelium of the organism penetrates all parts of the leaves. Pseudothecia are partially formed during autumn and early winter, and they will remain dormant until spring. At this time growth is resumed and the life-cycle is again started (Hesler 1917). Pathogenesis is visualized in Figure 1. Figure 2 shows infection at the later stage of development of scab on fruits and leaves. 3 Chapter 1 Figure 1. Life cycle of V. inaequalis (reprinted from Agrios 1998) 2a 2b Figure 2. 2a. Scab infected fruits, 2b. Scab infected leaf Genetic improvement of apple Most of present day cultivars are susceptible to apple scab disease and fruit growers have to spray 15 times with fungicides in a season. Wild species of Malus are resistant to the scab disease (Soriano et al. 2009) and can be good sources of transfer of scab resistance to high quality susceptible cultivars.