Roberta Paris

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.

Genetic Diversity, Population Structure and Construction of a Core Collection of Apple Cultivars from Italian Germplasm

Apple germplasm collections are increasingly appreciated as a repository for the genetic improvement of species, and their evaluation is an essential prerequisite for their utilization in apple breeding. A set of 418 apple genotypes, including 383 accessions from the Italian germplasm and 35 International cultivars as reference, was analyzed using 15 SSRs with the aim of assessing the genetic diversity within this panel of varieties, evaluating relationships among them and determining their genetic structure. Genetic analyses performed by Bayesian model-based clustering revealed a clear differentiation of two major groups (G1 and G2). Local Italian accessions were grouped mainly in G2 while all except one of the reference cultivars were found in G1. Each of these two clusters has been further divided into two subgroups by a nested approach. These results were confirmed by factorial correspondence (FCA) and molecular variance (AMOVA) analyses. A core collection of 55 accessions, representative of the Italian apple germplasm and capable of retaining all the 238 SSR alleles detected on 192 unique genotypes, was established by the M-strategy method. The Italian apple germplasm represents an important source of genetic diversity which can be used, in addition to other characterized European germplasm collections, to optimize the efficiency of genome-wide association studies aimed at identifying the genomic regions controlling major horticultural traits.

Identification of functional apple scab resistance gene promoters.

Apple scab (Venturia inaequalis) is one of the most damaging diseases affecting commercial apple production. Some wild Malus species possess resistance against apple scab. One gene, HcrVf2, from a cluster of three genes derived from the wild apple Malus floribunda clone 821, has recently been shown to confer resistance to apple scab when transferred into a scab-susceptible apple variety. For this proof-of-function experiment, the use of the 35S promoter from Cauliflower mosaic virus was reliable and appropriate. However, in order to reduce the amount of non-plant DNA in genetically modified apple to a minimum, with the aim of increasing genetically modified organism acceptability, these genes would ideally be regulated by their own promoters. In this study, sequences from the promoter region of the three members of the HcrVf gene family were compared. Promoter constructs containing progressive 5′ deletions were prepared and used for functional analyses. Qualitative assessment confirmed promoter activity in apple. Quantitative promoter comparison was carried out in tobacco (Nicotiana glutinosa) and led to the identification of several promoter regions with different strengths from a basal level to half the strength of the 35S promoter from Cauliflower mosaic virus.

Simulated environmental criticalities affect transglutaminase of Malus and Corylus pollens having different allergenic potential

Increases in temperature and air pollution influence pollen allergenicity, which is responsible for the dramatic raise in respiratory allergies. To clarify possible underlying mechanisms, an anemophilous pollen (hazel, Corylus avellana), known to be allergenic, and an entomophilous one (apple, Malus domestica), the allergenicity of which was not known, were analysed. The presence also in apple pollen of known fruit allergens and their immunorecognition by serum of an allergic patient were preliminary ascertained, resulting also apple pollen potentially allergenic. Pollens were subjected to simulated stressful conditions, provided by changes in temperature, humidity, and copper and acid rain pollution. In the two pollens exposed to environmental criticalities, viability and germination were negatively affected and different transglutaminase (TGase) gel bands were differently immunodetected with the polyclonal antibody AtPng1p. The enzyme activity increased under stressful treatments and, along with its products, was found to be released outside the pollen with externalisation of TGase being predominant in C. avellana, whose grain presents a different cell wall composition with respect to that of M. domestica. A recombinant plant TGase (AtPng1p) stimulated the secreted phospholipase A2 (sPLA2) activity, that in vivo is present in human mucosa and is involved in inflammation. Similarly, stressed pollen, hazel pollen being the most efficient, stimulated to very different extent sPLA2 activity and putrescine conjugation to sPLA2. We propose that externalised pollen TGase could be one of the mediators of pollen allergenicity, especially under environmental stress induced by climate changes.

Genomic organisation of the Mal d 1 gene cluster on linkage group 16 in apple

European populations exhibit progressive sensitisation to food allergens, and apples are one of the foods for which sensitisation is observed most frequently. Apple cultivars vary greatly in their allergenic characteristics, and a better understanding of the genetic basis of low allergenicity may therefore allow allergic individuals to increase their fruit intake. Mal d 1 is considered to be a major apple allergen, and this protein is encoded by the most complex allergen gene family. Not all Mal d 1 members are likely to be involved in allergenicity. Therefore, additional knowledge about the existence and characteristics of the different Mal d 1 genes is required. In the present study, we investigated the genomic organisation of the Mal d 1 gene cluster in linkage group 16 of apple through the sequencing of two bacterial artificial chromosome clones. The results provided new information on the composition of this family with respect to the number and orientation of functional and pseudogenes and their physical distances. The results were compared with the apple and peach genome sequences that have recently been made available. A broad analysis of the whole apple genome revealed the presence of new genes in this family, and a complete list of the observed Mal d 1 genes is supplied. Thus, this study provides an important contribution towards a better understanding of the genetics of the Mal d 1 family and establishes the basis for further research on allelic diversity among cultivars in relation to variation in allergenicity.

Mapping and functional analysis of four apple receptor-like protein kinases related to LRPKm1 in HcrVf2-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.

Cloning and expression of the major allergen genes in apple fruit

Summary Apple, the most common fruit in European and North American diets, can cause allergic reactions in susceptible individuals, and genes for four families of apple allergens have been identified to date: Mal d 1, Mal d 2, Mal d 3, and Mal d 4. Our remit was to evaluate the effects of genotype, tissue, and stage of fruit development on the expression of these allergen genes and, hence, on the potential allergenicity of apple fruit. Transcript levels were investigated in fruit of the apple cultivars, ‘Gala’ and ‘Florina’, using quantitative Real-Time PCR. The resulting patterns of allergen gene expression differed, with Mal d 1 and Mal d 2 being the most highly expressed in the skin and flesh of ripe fruit, respectively. Overall, ‘Florina’ fruit showed higher levels of expression than ‘Gala’ fruit for all allergens tested.

dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model

AbstractcDNA-AFLP analysis for transcript profiling has been successfully applied to study many plant biological systems, particularly plant–microbe interactions. However, the separation of cDNA-AFLP fragments by gel electrophoresis is reported to be labor-intensive with only limited potential for automation, and the collection of differential bands for gene identification is even more cumbersome. In this work, we present the use of dHPLC (denaturing high performance liquid chromatography) and automated DNA fragment collection using the WAVE® System to analyze and recover cDNA-AFLP fragments. The method is successfully applied to the Malus–Venturia inaequalis interaction, making it possible to collect 66 different transcript-derived fragments for apple genes putatively involved in the defense response activated by the HcrVf2 resistance gene. The results, validated by real time quantitative RT-PCR, were consistent with the plant–pathogen interaction under investigation and this further supports the suitability of dHPLC for cDNA-AFLP transcript profiling. Features and advantages of this new approach are discussed, evincing that it is an almost fully automatable and cost-effective solution for processing large numbers of samples and collecting differential genes involved in other biological processes and non-model plants.

Not the one, but the only one: about Cannabis cryptic virus in plants showing ‘hemp streak’ disease symptoms

Interveinal chlorosis and leaf margin wrinkling are widespread symptoms of Cannabis sativa. They are traditionally attributed to the so-called hemp streak virus (HSV), but its existence has not been demonstrated yet. To our knowledge, no molecular investigation has so far been performed in order to identify the causal agent of this symptomatology, we therefore decided to use traditional and molecular virology techniques to better characterize symptoms and pursue the etiological agent. No pathogenic virus was found by using targeted PCR reactions and by RNA sequencing, whereas we were able to detect the Cannabis cryptic virus (CanCV) with both techniques. We, therefore, developed an RT-qPCR assay based on a CanCV-specific TaqMan probe and applied it to a wide range of symptomatic and symptomless plants, using a two-step (for quantification), or a one-step (for fast detection) protocol. Both symptoms and the virus were only shown to be transmitted vertically and did not pass via mechanical inoculation or grafting, though we could not find any cause-effect correlation between them. In fact, the virus was found in all the tested hemp samples, and its abundance varied greatly between different accessions and individuals, independently from the presence and severity of symptoms. The suggestion that hemp streak is caused by a virus is therefore questioned. Some abiotic stresses seem to play a role in triggering the symptoms but this aspect needs further investigation. For breeding purposes, a selection of parental plants based on the absence of symptoms proved to be efficient in containment of the disease.

Erratum to: A qRT-PCR assay for the expression of all Mal d 1 isoallergen genes

Author details Wageningen UR Plant Breeding, Plant Research International, Droevendaalsesteeg 1, Wageningen PB 6708, The Netherlands. Department of Fruit Tree and Woody Plant Sciences, University of Bologna, Viale Fanin 46, Bologna 40127, Italy. Department of Paediatrics, University of Bologna, Via Massarenti 11, Bologna 40138, Italy. Present address: Consiglio per la Ricerca e la sperimentazione in Agricoltura-Centro di Ricerca per le Colture Industriali, via di Corticella 133, Bologna 40128, Italy.