R. Groenwold

Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (PMdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’.

Cloning and functional characterization of the Rvi15 (Vr2) gene for apple scab resistance

Apple scab, caused by Venturia inaequalis, is a serious disease of apple. Previously, the scab resistance Rvi15 (Vr2) from the accession GMAL 2473 was genetically mapped, and three candidate resistance genes were identified. Here, we report the cloning and functional characterization of these three genes, named Vr2-A, Vr2-B, and Vr2-C. Each gene was cloned with its native promoter, terminator and introns, and inserted into the susceptible apple cultivar ‘Gala’. Inoculation of the plants containing Vr2-A and Vr2-B induced no resistance symptoms, but abundant sporulation. However, inoculation of the plants harboring Vr2-C showed a hypersensitive response with clear pinpoint pits, and no or very little sporulation. We conclude that Vr2-C is the Rvi15 (Vr2) gene. This gene belongs to the Toll and mammalian interleukin-1 receptor protein nucleotide-binding site leucine-rich repeat structure resistance gene family. The proteins of this gene family reside in the cytoplasm, whereas V. inaequalis develops in the apoplast, between the epidermis and cuticle, without making haustoria. The spatial separation of the recognizing resistance protein and the pathogen is discussed. This is the second cloned gene for apple scab resistance, and out of these two the only one leading to a symplastic protein.

Cisgenic apple trees; development, characterization and performance

Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.

Virulence Characterization of Venturia inaequalis Reference Isolates on the Differential Set of Malus Hosts

A set of differential hosts has recently been identified for 17 apple scab resistance genes in an updated system for defining gene-for-gene (GfG) relationships in the Venturia inaequalis-Malus pathosystem. However, a set of reference isolates characterized for their complementary avirulence alleles is not yet available. In this paper, we report on improving the set of differential hosts for h(7) and propose the apple genotype LPG3-29 as carrying the single major resistance gene Rvi7. We characterized a reference set of 23 V. inaequalis isolates on 14 differential apple hosts carrying major resistance genes under controlled conditions. We identified isolates that were virulent on at least one of the following defined resistance gene hosts: h(1), h(2), h(3), h(4), h(5), h(6), h(7), h(8), h(9), h(10), and h(13). Sixteen different virulence patterns were observed. In general, the isolates carried one to three virulences, but some of them were more complex, with up to six virulences. This set of well-characterized isolates will be helpful for the identification of additional apple scab resistance genes in apple germplasm and the characterization of new GfG relationships to help improve our understanding of the host-pathogen interactions in the V. inaequalis-Malus pathosystem.