Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Apr;75(6):579-91.
doi: 10.1007/s11103-011-9749-1. Epub 2011 Feb 4.

Functional Analysis and Expression Profiling of HcrVf1 and HcrVf2 for Development of Scab Resistant Cisgenic and Intragenic Apples

Affiliations
Free PMC article

Functional Analysis and Expression Profiling of HcrVf1 and HcrVf2 for Development of Scab Resistant Cisgenic and Intragenic Apples

Sameer G Joshi et al. Plant Mol Biol. .
Free PMC article

Abstract

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 (P(MdRbc)) 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'.

Figures

Fig. 1
Fig. 1
Constructs used for plant transformation. P Promoter, cds coding sequence, T terminator. The numbers in parentheses indicate the lengths of promoter, coding sequence and terminator in basepairs. Vertical stripes represent apple rubisco promoter and horizontal stripes represent apple rubisco terminator. All LP and SP constructs represent stretches cloned as a whole; the PMdRbc constructs represent new combinations
Fig. 2
Fig. 2
Amplification of full length HcrVf genes by PCR from BAC clones 12 (HcrVf1) and 105 (HcrVf2). M = 1 Kb + DNA ladder, SPHcrVf1 = Short promoter HcrVf1, LPHcrVf1 = Long promoter HcrVf1, SPHcrVf2 = Short promoter HcrVf2, LPHcrVf2 = Long promoter HcrVf2
Fig. 3
Fig. 3
Estimation of transgene copy number in apple transformants through Southern hybridization. Probe: nptII; digestion by BglII. M = 1 kb + DNA ladder, + = positive control (plasmid), − = negative control (untransformed ‘Gala’)
Fig. 4
Fig. 4
a and b Relative expression of HcrVf genes under control of different gene promoters. SP short native promoter, LP long native promoter, PMdRbc apple rubisco promoter. Santana as reference was set at 1 (see also Table 2). For visual convenience two scales have been plotted together. Two oblique lines indicate change in the scale in the vertical axis
Fig. 5
Fig. 5
Sporulation of Vf avirulent monoconidial isolate EU-B05 of V. inaequalis as observed on leaves of HcrVf transgenic lines and of ‘Santana’ (resistant control) and of ‘Gala’ (susceptible control). The difference in the size of the leaves among the different plants with or without sporulation may be attributed to the improper development of leaves due to V. inaequalis growth

Similar articles

See all similar articles

Cited by 20 articles

See all "Cited by" articles

References

    1. Bäumelein H, Boerjan W, Nagy I, Panitz R, Inze D, Wobus U. Upstream sequences regulating legumin gene expression in heterologous transgenic plants. Mol Gen Genet. 1991;225:121–128. - PubMed
    1. Belfanti E, Silfverberg-Dilworth E, Tartarini S, Patocchi A, Barbieri M, Zhu J, Vinatzer BA, Gianfranceschi L, Gessler C, Sansavini S. The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. Proc Natl Acad Sci USA. 2004;101:886–890. doi: 10.1073/pnas.0304808101. - DOI - PMC - PubMed
    1. Bus VGM, Rikkerink EHA, Weg WE, Rusholme RL, Gardiner SE, Bassett HCM, Kodde LP, Parisi L, Laurens FND, Meulenbroek EJ, Plummer KM. The Vh2 and Vh4 scab resistance genes in two differential hosts derived from Russian Apple R12740–7A map to the same linkage group of apple. Mol Breeding. 2005;15:103–116. doi: 10.1007/s11032-004-3609-5. - DOI
    1. Cervera M, Pina JA, Navarro L, Peña L. A broad exploration of transgenic population of citrus: stability of gene expression and phenotype. Theor Appl Genet. 2000;100:670–677. doi: 10.1007/s001220051338. - DOI
    1. Chevalier M, Lespinasse Y, Renaudin S. A microscopy study of different classes of symptoms coded by the Vf gene in apple for resistance to scab (Venturia inaequalis) Plant Pathol. 1991;40:249–256. doi: 10.1111/j.1365-3059.1991.tb02374.x. - DOI

Publication types

MeSH terms

LinkOut - more resources

Feedback