The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety

Abstract

The Vf gene from the wild species Malus floribunda 821 is the most studied apple scab resistance gene. Several molecular markers mapping around this gene were the starting point for a positional cloning project. The analysis of the bacterial artificial chromosome clones spanning the Vf region led to the identification of a cluster of genes homologous to the Cladosporium fulvum resistance gene family of tomato. One of these genes, HcrVf2 (homologue of the C. fulvum resistance genes of the Vf region), was used to transform the susceptible apple cultivar Gala. Four independent transformed lines resistant to apple scab were produced, proving that HcrVf2 is sufficient to confer scab resistance to a susceptible cultivar. The results show that direct gene transfer between cross-compatible species can be viable when, as in apple, the use of backcrosses to introduce resistance genes from wild species cannot exactly reconstitute the heterozygous genotype of clonally propagated cultivars.

Fig. 1.

(a) Schematic representation of T-DNA from pCORF2 used in the transformation experiment. nptII and the putative scab resistance gene HcrVf2 are regulated by the cauliflower mosaic virus 35S promoters (filled arrows). E, EcoRI; B, BstEII; H, HindIII; LB, left border; RB, right border; CaMV, cauliflower mosaic virus 35S poly(A) signal; Nos, Nos poly(A) signal. H¹, H², and H³ represent restriction sites 1, 2, and 3, respectively, and thin bars represent the distance between HindIII restriction sites. Thick bars represent Southern blot probes. (b) T-DNA integration and copy number. Southern blot hybridization of HindIII-digested genomic DNA. Lanes: 1, Ga2-7; 2, Ga2-2; 3, Gala; 4, Ga2-5; 5, Ga2-21; 6, Gala; 7, Ga2-8. (Upper) T-DNA was hybridized with HcrVf2 probe, showing, in addition to the 1.2-kb specific fragment of the HcrVf2 gene, a second fragment for the estimation of the copy number. [The 300-bp fragment produced by HindIII sites 2 and 3 (H² and H³ in a) was too small and was not transferred to the membrane.] Ga2-2, Ga2-5, Ga2-8, and Ga2-21 each contain a single copy of the pCORF2 T-DNA. Ga2-7 contains no HcrVf2 gene. T-DNA integration is shown by fragments of 9.0, 4.4, 3.0, and 2.4 kb hybridizing to this probe for the lines Ga2-2, Ga2-5, Ga2-8, and Ga2-21, respectively. (Lower) nptII probe. All transgenic lines show one single hybridization band of specific size demonstrating the presence of and integration to the nptII gene.

Fig. 2.

Transcription evaluated by RT-PCR. Lanes: 1,100-bp ladder; 2, Enterprise; 3, Florina; 4, Gala; 5, Ga2-2; 6, Ga2-5; 7, Ga2-8; 8, Ga2-21; 9, Ga2-7. (a) HcrVf2 primers show that the gene is expressed in Vf cvs. and in all transformed lines except Ga2-7. (b) nptII primers show that the gene is expressed only in transformed lines.

Fig. 3.

Leaf symptoms 21 days after inoculation with V. inaequalis conidia. Transgenic line Ga2-21 (Upper Right), containing HcrVf2, represents typical symptoms observed on all HcrVf2 transformed lines. Transgenic line Ga2-7 (Lower Right) contains only the selective nptII gene and is thus a transgenic control. The susceptible cv. Gala (Lower Left) and resistant cv. Florina (Upper Left) are included for the comparison of scab symptoms.