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

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'.

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 P_MdRbc constructs represent new combinations

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

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

a and b Relative expression of HcrVf genes under control of different gene promoters. SP short native promoter, LP long native promoter, P _MdRbc 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

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