Scab resistance in ‘Geneva’ apple is conditioned by a resistance gene cluster with complex genetic control

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most severe diseases of apple worldwide. It is the most studied plant–pathogen interaction involving a woody species using modern genetic, genomic, proteomic and bioinformatic approaches in both species. Although ‘Geneva’ apple was recognized long ago as a potential source of resistance to scab, this resistance has not been characterized previously. Differential interactions between various monoconidial isolates of V. inaequalis and six segregating F1 and F2 populations indicate the presence of at least five loci governing the resistance in ‘Geneva’. The 17 chromosomes of apple were screened using genotyping‐by‐sequencing, as well as single marker mapping, to position loci controlling the V. inaequalis resistance on linkage group 4. Next, we fine mapped a 5‐cM region containing five loci conferring both dominant and recessive scab resistance to the distal end of the linkage group. This region corresponds to 2.2 Mbp (from 20.3 to 22.5 Mbp) on the physical map of ‘Golden Delicious’ containing nine candidate nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) resistance genes. This study increases our understanding of the complex genetic basis of apple scab resistance conferred by ‘Geneva’, as well as the gene‐for‐gene (GfG) relationships between the effector genes in the pathogen and resistance genes in the host.     

Genetics of resistance in apple against <Emphasis Type="Italic">Venturia inaequalis</Emphasis> (Wint.) Cke

Apple (Malus × domestica) is the third important fruit in terms of production and consumption worldwide. Apple scab caused by Venturia inaequalis is the most devastating disease of apple. In the apple-growing regions, many fungicides are sprayed to control the disease leading to increase in the production cost. Development of scab-resistant cultivars is the long-lasting solution to control the disease. In apples, more than 20 major scab resistance genes have been identified in various cultivars and few wild relatives. Of all these genes, Rvi6 derived from Malus floribunda has been most extensively used in different breeding programs. Gene for gene interactions of these resistance genes with the avirulence genes from V. inaequalis have been understood in many cases. QTL-based polygenic resistance has also been characterized in apple. Nucleotide Binding Site Leucine-Rich Repeats (NBS-LRR) have been identified from the apple genome and many of them have been characterized from the scab resistance region. Molecular markers associated with most of the major scab resistance genes have been identified and their position has been mapped on different linkage groups. Marker-assisted selection (MAS) can be helpful in speeding up and accurately identifying the scab-resistant parents and progeny. Pyramiding of several major resistance genes can be undertaken for more durable resistance against apple scab. The present paper reviews the Malus-Venturia pathosystem, current status of knowledge about scab resistance genes, and their application in breeding against apple scab.     

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.     

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. JMS and SGJ contributed equally to this work     

Mapping and functional analysis of four apple receptor-like protein kinases related to <Emphasis Type="Italic">LRPKm1</Emphasis> in <Emphasis Type="Italic">HcrVf2</Emphasis>-transgenic and wild-type apple plants

The Malus–Venturia inaequalis interaction is the most studied plant–pathogen interaction involving a woody species. Besides the cloning of an apple scab resistance gene HcrVf2, several sequences have been recently identified that are modulated after pathogen recognition in Vf-resistant genotypes. Among these, there is a putative leucine-rich repeat receptor-like protein kinase from the apple scab-resistant cv. Florina, named LRPKm1 that is induced after V. inaequalis inoculation and salicylic acid treatment. In this work, the isolation, characterization, and mapping of four new genes belonging to the LRPKm multigene family are reported. According to their cumulative expression profiles in HcrVf2-transgenic and wild-type apple plants treated with V. inaequalis, LRPKm genes have been divided in two groups. LRPKm1 and LRPKm3, giving a response related to the presence of HcrVf2, are probably involved in the recognition of pathogen-derived signals. LRPKm2 and LRPKm4, with an expression profile unrelated to the HcrVf2 gene, are putatively involved in the plant basal defense. Furthermore, we have localized LRPKm proteins at the cytological level in the plasma membrane of epidermal cells in resistant genotypes following pathogen challenge, thus confirming software predictions and molecular results. The possible involvement of LRPKm proteins in apple scab resistance and in the plant basal defense makes them attractive for a better comprehension of the molecular mechanisms of the signal transduction pathways after pathogen recognition.     

Identification of a leucine-rich repeat receptor-like serine/threonine-protein kinase as a candidate gene for <Emphasis Type="Italic">Rvi12 (Vb)</Emphasis>-based apple scab resistance

Apple scab caused by Venturia inaequalis is the most important fungal disease of apples (Malus × domestica). Currently, the disease is controlled by up to 15 fungicide applications to the crop per year. Resistant apple cultivars will help promote the sustainable control of scab in commercial orchards. The breakdown of the Rvi6 (Vf) major-gene based resistance, the most used resistance gene in apple breeding, prompted the identification and characterization of new scab resistance genes. By using a large segregating population, the Rvi12 scab resistance gene was previously mapped to a genetic location flanked by molecular markers SNP_23.599 and SNP_24.482. Starting from these markers, utilizing chromosome walking of a Hansen’s baccata #2 (HB2) BAC-library; a single BAC clone spanning the Rvi12 interval was identified. Following Pacific Biosciences (PacBio) RS II sequencing and the use of the hierarchical genome assembly process (HGAP) assembly of the BAC clone sequence, the Rvi12 resistance locus was localized to a 62.3-kb genomic region. Gene prediction and in silico characterization identified a single candidate resistance gene. The gene, named here as Rvi12_Cd5, belongs to the LRR receptor-like serine/threonine-protein kinase family. In silico comparison of the resistance allele from HB2 and the susceptible allele from Golden Delicious (GD) identified the presence of an additional intron in the HB2 allele. Conserved domain analysis identified the presence of four additional LRR motifs in the susceptible allele compared to the resistance allele. The constitutive expression of Rvi12_Cd5 in HB2, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in apple.     

Expression profiling in HcrVf2-transformed apple plants in response to Venturia inaequalis

Apple scab resistance is one of the most well-characterized plant–pathogen interactions in a woody plant species. While the HcrVf2 gene from the wild apple Malus floribunda 821 has proved capable of conferring scab resistance to the susceptible cv. Gala after genetic transformation, its identification represents only the first step in understanding the molecular mechanisms and, hence, the network of genes underlying the defence response. We used a PCR-based suppression subtractive hybridization to identify apple genes that are differentially expressed after Venturia inaequalis inoculation. Subtractive hybridization was performed between cDNA from challenged leaves of HcrVf2-resistant transgenic Gala and susceptible cv. Gala plants. A library of 523 unigenes was constructed and characterized by assigning a putative function via comparison with public databases. This set of pathogen-modulated apple genes includes many defence-related genes and is therefore an important source of information for understanding the molecular basis of the Malus–V. inaequalis interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two receptor-like genes, Vfa1 and Vfa2, confer resistance to the fungal pathogen Venturia inaequalis inciting apple scab disease

The Vf locus, originating from the crabapple species Malus floribunda 821, confers resistance to five races of the fungal pathogen Venturia inaequalis, the causal agent of apple scab disease. Previously, a cluster of four receptor-like genes, Vfa1, Vfa2, Vfa3, and Vfa4, was identified within the Vf locus. Because the amino-acid sequence of Vfa3 is truncated, it was deemed nonfunctional. In this study, each of the three full-length Vfa genes was introduced into a plant cloning vector, pCAMBIA2301, and used for Agrobacterium-mediated transformation of two apple cultivars, Galaxy and McIntosh, to assess functionality of these genes and to characterize their roles in resistance to V. inaequalis. Transformed apple lines carrying each of Vfa1, Vfa2, or Vfa4 were developed, analyzed for the presence of the transgene using polymerase chain reaction and Southern blotting, and assayed for resistance to apple scab following inoculation with V. inaequalis. Transformed lines expressing Vfa4 were found to be susceptible to apple scab, whereas those expressing either Vfa1 or Vfa2 exhibited partial resistance to apple scab. Based on Western blot analysis as well as microscopic analysis of plant resistance reactions, the roles of Vfa1 and Vfa2 in apple scab disease resistance response are discussed.     

Cellulolytic Ability of the Scab Fungus, Venturia inaequalis

Cellulase and β-D-glucosidase activity have been identified in Venturia inaequalis, the causal agent of apple scab. Most of the activity of the enzymes was associated with the mycelium after homogenization. The activities were relatively weak in vitro compared to those of other plant pathogens. With a new screening technique, β-D-glucosidase hyperproducing mutants were obtained.     

Molecular markers linked to the apple scab resistance gene <Emphasis Type="Italic">Vbj</Emphasis> derived from <Emphasis Type="Italic">Malus baccata jackii</Emphasis>

Breeding for scab-resistant apple cultivars by pyramiding several resistance genes in the same genetic background is a promising way to control apple scab caused by the fungus Venturia inaequalis. To achieve this goal, DNA markers linked to the genes of interest are required in order to select seedlings with the desired resistance allele combinations. For several apple scab resistance genes, molecular markers are already available; but until now, none existed for the apple scab resistance gene Vbj originating from the crab apple Malus baccata jackii. Using bulk segregant analysis, three RAPD markers linked to Vbj were first identified. These markers were transformed into more reliable sequence-characterised amplified region (SCAR) markers that proved to be co-dominant. In addition, three SSR markers and one SCAR were identified by comparing homologous linkage groups of existing genetic maps. Discarding plants showing genotype–phenotype incongruence (GPI plants) plants, a linkage map was calculated. Vbj mapped between the markers CH05e03 (SSR) and T6-SCAR, at 0.6 cM from CH05e03 and at 3.9 cM from T6-SCAR. Without the removal of the GPI plants, Vbj was placed 15 cM away from the closest markers. Problems and pitfalls due to GPI plants and the consequences for mapping the resistance gene accurately are discussed. Finally, the usefulness of co-dominant markers for pedigree analysis is also demonstrated.     

Functional analysis and expression profiling of <Emphasis Type="Italic">HcrVf1</Emphasis> and <Emphasis Type="Italic">HcrVf2</Emphasis> 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 (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’.     

How Did Host Domestication Modify Life History Traits of Its Pathogens?

Understanding evolutionary dynamics of pathogens during domestication of their hosts and rise of agro-ecosystems is essential for durable disease management. Here, we investigated changes in life-history traits of the fungal pathogen Venturia inaequalis during domestication of the apple. Life traits linked to fungal dispersal were compared between 60 strains that were sampled in domestic and wild habitats in Kazakhstan, the center of origin of both host and pathogen. Our two main findings are that transition from wild to agro-ecosystems was associated with an increase of both spore size and sporulation capacity; and that distribution of quantitative traits of the domestic population mostly overlapped with those of the wild population. Our results suggest that apple domestication had a considerable impact on fungal characters linked to its dispersal through selection from standing phenotypic diversity. We showed that pestification of V. inaequalis in orchards led to an enhanced allocation in colonization ability from standing variation in the wild area. This study emphasizes the potential threat that pathogenic fungal populations living in wild environments represent for durability of resistance in agro-ecosystems.     

Interaction between Venturia inaequalis and Apple Callus Tissue Cultures: an Electron Microscopic Study

Untrastructural interactions between Venturia inaequalis and callus cultures from scab susceptible and resistant apple varieties, were similar. Host cell wall changes, appositions, and invagination of host plasmamembrane at sites of close contact with fungal hyphae were regularly observed. The ultrastructural observations are described and discussed. The host cell alterations as well as many fungal structures corresponded to those known in young leaves of susceptible apple varieties.