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. <br/

Spherical Dust Collapse in Higher Dimensions

We consider here the question if it is possible to recover cosmic censorship when a transition is made to higher dimensional spacetimes, by studying the spherically symmetric dust collapse in an arbitrary higher spacetime dimension. It is pointed out that if only black holes are to result as end state of a continual gravitational collapse, several conditions must be imposed on the collapsing configuration, some of which may appear to be restrictive, and we need to study carefully if these can be suitably motivated physically in a realistic collapse scenario. It would appear that in a generic higher dimensional dust collapse, both black holes and naked singularities would develop as end states as indicated by the results here. The mathematical approach developed here generalizes and unifies the earlier available results on higher dimensional dust collapse as we point out. Further, the dependence of black hole or naked singularity end states as collapse outcomes, on the nature of the initial data from which the collapse develops, is brought out explicitly and in a transparent manner as we show here. Our method also allows us to consider here in some detail the genericity and stability aspects related to the occurrence of naked singularities in gravitational collapse.Comment: Revtex4, Title changed, To appear in Physical Review

Development of cisgenic apples with durable resistance to apple scab

Most of the apple (Malus × domestica) growers are facing serious disease problems with apple scab which is caused by the fungus Venturia inaequalis. Developing a resistant variety in apple through classical breeding is very slow and inefficient. So, we aim at improving existing apple varieties through a new concept called “cisgenesis” which saves time and effort compared to classical breeding. Malus floribunda proved to be a good source of natural scab resistance genes. The genes HcrVf1 and HcrVf2, consisting of promoter, coding and terminator sequences in their natural configuration, were isolated from Malus floribunda and cloned into the binary vector pMF1. Apple cv. ‘Gala’ was transformed with pMF1 containing HcrVf1 and HcrVf2, individually or in combination. pMF1 can be used to obtain marker-free plants by recombinase-based excision of a fragment carrying undesired gene sequences, such as antibiotic-selection marker genes, leaving behind only the gene(s)-of-interest and one recombination site. Using this vector it is therefore possible to stack several genes by retransformation using the same selection procedure. In order to obtain durable resistance, we have the intention to combine different resistance genes from Malus either by stacking them one by one or by introducing them all together in one T-DNA. Performance of all different types of cisgenic plants will be evaluated by monitoring scab resistance levels phenotypically and by determining gene expression profiles through quantitative RT-PC

Identification and mapping of a new apple scab resistance gene

Here we report the identification of a new resistance gene (Vd3) against apple scab (Venturia inaequalis) from the apple selection 1980-015-25 of the breeding program at Plant Research International. This accession also contains the Vf gene. We mapped Vd3, using SSR and DArT markers, on linkage group 1, at a distance of 6 cM from Vf gene, but in repulsion phase to Vf. Based on pedigree analysis and resistance tests, it could be deduced that 1980-015-25 had inherited Vd3 from the founder D3. This gene provides resistance to the highly virulent EU-NL-24 strain of the race 7 of V. inaequalis. This strain has overcome the resistance from both Vf and Vg. However, Vd3 has been not effective against the majority of other V. inaequalis strains we used in our disease test

Vibration Isolation and Transmissibility Characteristics of Passive Sequential Damper

This paper presents a half-car model (4-degrees-of-freedom) employing nonlinear passlve sequential damper. The vibration isolation and transmrssibility effect on the vehicle's centre ofgravity (C.G.) has been studied. The results have been compared for transmissibility, displacement, and velocity transient response for half-car model having nonlinear passive sequentialhydropneumatic damper under different terrain excitation

Identification and mapping of the novel apple scab resistance gene Vd3

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing in temperate zones with humid springs and summers. Breeding programs around the world have been able to identify several sources of resistance, the Vf from Malus floribunda 821 being the most frequently used. The appearance of two new races of V. inaequalis (races 6 and 7) in several European countries that are able to overcome the resistance of the Vf gene put in evidence the necessity of the combination of different resistance genes in the same genotype (pyramiding). Here, we report the identification and mapping of a new apple scab resistance gene (Vd3) from the resistant selection “1980-015-25” of the apple breeding program at Plant Research International, The Netherlands. This selection contains also the Vf gene and the novel V25 gene for apple scab resistance. We mapped Vd3 on linkage group 1, 1 cM to the south of Vf in repulsion phase to it. Based on pedigree analysis and resistance tests, it could be deduced that 1980-015-25 had inherited Vd3 from the founder “D3.” This gene provides resistance to the highly virulent EU-NL-24 strain of race 7 of V. inaequalis capable of overcoming the resistance from Vf and Vg

Higher dimensional dust collapse with a cosmological constant

The general solution of the Einstein equation for higher dimensional (HD) spherically symmetric collapse of inhomogeneous dust in presence of a cosmological term, i.e., exact interior solutions of the Einstein field equations is presented for the HD Tolman-Bondi metrics imbedded in a de Sitter background. The solution is then matched to exterior HD Scwarschild-de Sitter. A brief discussion on the causal structure singularities and horizons is provided. It turns out that the collapse proceed in the same way as in the Minkowski background, i.e., the strong curvature naked singularities form and that the higher dimensions seem to favor black holes rather than naked singularities.Comment: 7 Pages, no figure

Why do naked singularities form in gravitational collapse?

We investigate what are the key physical features that cause the development of a naked singularity, rather than a black hole, as the end-state of spherical gravitational collapse. We show that sufficiently strong shearing effects near the singularity delay the formation of the apparent horizon. This exposes the singularity to an external observer, in contrast to a black hole, which is hidden behind an event horizon due to the early formation of an apparent horizon.Comment: revised for clarity, new figure included; version accepted by Phys. Rev. D (RC

Gravitational collapse of null fluid on the brane

We first obtain the analogue of Vaidya's solution on the brane for studying the collapse of null fluid onto a flat Minkowski cavity on the brane. Since the back-reaction of the bulk onto the brane is supposed to strengthen gravity on the brane, it would favour formation of black hole as against naked singularity. That is the parameter window in the initial data set giving rise to naked singularity in the 4D Vaidya case would now get partially covered.Comment: 12 pages, latex, 3 figures, submitted to Phys.Lett.