Towards durabale resistance to apple scab using cisgenes

Apple (Malus x domestica) is one of the important fruit crops of the world. It is mainly cultivated in temperate regions. Apple fruit contains many health beneficial compounds which may play an important role in reducing cancer cell proliferation and lowering the level of cholesterol. Apple production can suffer from several pests and diseases and among them scab is very important. Apple scab is a fungal disease caused by Venturia inaequalis. The pathogen is a facultative saprophyte that grows during the growing season subcuticularly on the host. Most of the present day high quality apple cultivars are susceptible to apple scab. The crop loss due to apple scab has been amount to more than 70%. Fruit growers usually spray fungicides 15 times or more in a season to control the scab disease. To reduce the use of chemicals, it is absolute necessary to develop apple varieties with durable scab resistance. Conventional breeding in apple has some drawbacks such as long generation period, genetic drag and the self-incompatible sexual reproduction system. Therefore, stacking of more than one resistance gene by classical introgression breeding is inefficient. Genetic modification is an alternative option to improve the existing scab-susceptible varieties into scab-resistant ones. However, consumer acceptance of transgenic food in Europe is a problem. Therefore, we developed a genetic modification system with cisgenes and intragenes instead of transgenes. Cisgenes are genes from the plant itself or from crossable species with their natural introns and own regulatory elements in normal sense orientation. Intragenes are like cisgenes containing only functional parts of genes from the plant itself or from crossable species, however, these functional parts originate from different genes. All these genes or gene parts are belonging to the normal breeder’s gene pool. Transgenes are synthetic genes or (partly) origination from non-crossable species, like viruses and microorganisms. Transgenes are representing a new gene pool for plant breeding. GMO-regulations have been developed for transgenes. Societal research showed that consumer preference for cisgenic food is higher than for transgenic food. Cisgenic or intragenic plants can be developed by transferring the desired scab resistant genes into the scab-susceptible cultivar through Agrobacterium tumefaciens-mediated transformation. Transformation aimed at cisgenesis or intragenesis should be done either without the use of selectable marker genes or by using selection markers first and eliminating them subsequently after selection of transformants. In this thesis almost all steps have been made to come to cisgenic apple plants with resistance to scab disease (chapter 2). Although many scab resistance genes have been identified and mapped, only Vf has been positionally cloned. Vf is a locus with four paralogs namely HcrVf1 (Homologues of Cladosporium fulvum resistance genes of Vf region), HcrVf2, HcrVf3, and HcrVf4. Only HcrVf1 and HcrVf2 are considered as being functional. In conventional breeding Vf inherits as a single locus so it is not possible to study the individual role of HcrVf1 and HcrVf2 in conferring resistance against scab using conventionally bred material. The present study was set up to study in depth the roles of HcrVf1 and HcrVf2 separately in conferring resistance to apple scab, using A. tumefaciens mediated transformation. Both isolated genes were regulated as cisgenes by their own promoter and terminator sequences. The two cisgenes were used in two different lengths of the 5’-upstream sequences, so with a short promoter (SP) and a long promoter (LP) i.e. 312 bp and 1990 bp for HcrVf1 and 288 bp and 2000 bp for HcrVf2. HcrVf1 and HcrVf2 were also combined with the apple rubisco promoter and terminator into intragenes because these regulatory elements were found to give high expression in plants. The HcrVf1 and HcrVf2 cisgenes and intragenes were inserted into the susceptible cv. ‘Gala’, using the marker free system pMF1. Several apple transformants were selected for further characterization. Micrografting was carried out in order to take the ‘in vitro’ transformants to the greenhouse. This method proved to promote growth better than rooting of ‘in vitro’ transformants. Apple transformant ‘in vitro’ shoots were used as scions and grafted onto the apple seedling rootstocks. Micrografts were ready for further testing 4 to 5 weeks after grafting. At this stage the young leaves were collected for isolation of DNA and RNA. Southern hybridization was performed to check the inserted T-DNA copy number. For this, the selection marker gene nptII was used as a probe. Most of the transformants (17) were found to have a single T-DNA insert and seven transformants showed two T-DNA inserts. Subsequently, HcrVf gene expression in transformed lines was studied through quantitative RT-PCR (qRT-PCR) in relation to the natural HcrVf expression in the resistant cv. ‘Santana’. In case of HcrVf1 transformants, expression by LP was significantly higher than by SP, while in HcrVf2 transformants no significant difference between SP and LP could be demonstrated. Both HcrVf1 and HcrVf2 genes showed highest expression when regulated by the apple rubisco promoter and terminator. Two HcrVf2 transformants, LPHcrVf2-4 and PMdRbcHcrVf2-12, showed the highest gene expression for the cisgene and intragene situation, respectively. Among HcrVf transformants, no significant correlation was observed between inserted gene copy number and gene expression level (Chapter 3). Micrografted cvs. ‘Santana’ (resistant control containing Vf through classical breeding), ‘Gala’ (susceptible control) and different micrografted apple transformants were tested for scab resistance against V. inaequalis isolate EU-B05. The top four leaves were Summary 125 used for inoculation with V. inaequalis. Seventeen days after inoculation, the plants were scored for sporulation using a quantitative scale. All the HcrVf1 transformants showed complete sporulation similar to the level in cv. ‘Gala’, indicating that HcrVf1 is not giving resistance. On the other hand, 10 out of the 13 HcrVf2 transformants showed resistance at levels that were statistically similar to cv. ‘Santana’. Two HcrVf2 transformants, LPHcrVf2-4 and PMdRbcHcrVf2-12, showed the best resistance. A negative correlation between HcrVf2 gene expression and sporulation was observed i.e. as gene expression increased there was a decrease in the fungal sporulation (Chapter 4). The results obtained by the scab experiment were used to select HcrVf1 and HcrVf2 transformants to check the resistance spectrum against different isolates of V. inaequalis. The plants were inoculated with four avirulent isolates of the pathogen and two isolates virulent to the resistant cv. ‘Santana’. The top two leaves were inoculated with fungal spores and the inoculated plants were scored for sporulation 21 days after inoculation. All the HcrVf1 transformants showed heavy sporulation of all the isolates used and they were behaving like untransformed cv. ‘Gala’. The HcrVf2 transformants were behaving like cv. ‘Santana’ indicating that the resistance coming from the Vf gene cluster is from HcrVf2 alone (Chapter 5). In order to increase the durability of resistance against scab, it is desired to stack several resistance genes into apple cultivars either by classical breeding or by genetic modification. To use it in a cisgenic or intragenic approach, new scab resistance genes have to be identified in apple and cloned. In chapter 6 it is described how a novel scab resistance gene, Vd3, has been identified and genetically mapped in the resistant selection “1980-015-025”. In the study we used the F1 progeny 2000-012 that is derived from the crossing between the resistant parent 1980-015-025 and the susceptible parent 1973-001-041. Mainly DArT markers were used in this genetic mapping study. Other known markers, such as SSRs, P-136 (RAPD marker), and Vf2ARD (RGA marker), were used for annotation of the linkage groups. The Vd3 gene has been mapped 1 cM to the south of the Vf gene cluster in repulsion phase on linkage group 1. Paternity tests have indicated that clone 1980-015-025 has inherited the Vd3 gene from founder accession D3. This gene can provide resistance against the virulent isolate EU-NL24, which can overcome the resistance of Vf and Vg. However, this gene cannot provide resistance against other isolates (Chapter 6). The results described in this thesis are of practical importance. Cisgenesis or intragenesis can be employed to provide multiple gene resistance against scab in apple without linkage drag problems as observed during classical introgression breeding. Our first potential cisgenic scab resistant ‘Gala’ plants with the HcrVf2 gene are being developed which can be used in regions free of virulent isolates. The cisgenic approach is essential in rapid improving a crop such as apple where it takes many decades through conventional breeding.