Insights into the molecular mechanisms underlying responses of apple trees to abiotic stresses

Apple(Malus×domestica)is a popular temperate fruit crop worldwide.However,its growth,productivity,and quality are often adversely affected by abiotic stresses such as drought,extreme temperature,and high salinity.Due to the long juvenile phase and highly heterozygous genome,the conventional breeding approaches for stress-tolerant cultivars are time-consuming and resource-intensive.These issues may be resolved by feasible molecular breeding techniques for apples,such as gene editing and marker-assisted selection.Therefore,it is necessary to acquire a more comprehensive comprehension of the molecular mechanisms underpinning apples’response to abiotic stress.In this review,we summarize the latest research progress in the molecular response of apples to abiotic stressors,including the gene expression regulation,protein modifications,and epigenetic modifications.We also provide updates on new approaches for improving apple abiotic stress tolerance,while discussing current challenges and future perspectives for apple molecular breeding.

Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine

Erysiphe necator is the causal agent of powdery mildew(PM),one of the most destructive diseases of grapevine.PM is controlled by sulfur-based and synthetic fungicides,which every year are dispersed into the environment.This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production.PM resistance can be achieved in other crops by knocking out susceptibility S-genes,such as those residing at genetic loci known as MLO(Mildew Locus O).All MLO S-genes of dicots belong to the phylogenetic clade V,including grapevine genes VvMLO7,11 and 13,which are upregulated during PM infection,and VvMLO6,which is not upregulated.Before adopting a gene-editing approach to knockout candidate S-genes,the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary.This paper reports the knockdown through RNA interference of VvMLO6,7,11 and 13.The knockdown of VvMLO6,11 and 13 did not decrease PM severity,whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%.The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance,whereas a role for VvMLO11 does not seem likely.Cell wall appositions(papillae)were present in both resistant and susceptible lines in response to PM attack.Thirteen genes involved in defense were less upregulated in infected mlo plants,highlighting the early mlo-dependent disruption of PM invasion.

The Arabidopsis pattern recognition receptor EFR enhances fire blight resistance in apple

Fire blight disease,caused by the bacterium Erwinia amylovora(E.amylovora),is responsible for substantial losses in cultivated apples worldwide.An important mechanism of plant immunity is based on the recognition of conserved microbial molecules,named pathogen-associated or microbe-associated molecular patterns(PAMPs or MAMPs),through pattern recognition receptors(PRRs),leading to pattern-triggered immunity(PTI).The interspecies transfer of PRRs represents a promising strategy to engineer broad-spectrum and durable disease resistance in crops.EFR,the Arabidopsis thaliana PRR for the PAMP elf18 derived from the elongation factor thermal unstable(EF-Tu)proved to be effective in improving bacterial resistance when expressed into Solanaceae and other plant species.In this study,we tested whether EFR can affect the interaction of apple with E.amylovora by its ectopic expression in the susceptible apple rootstock M.26.Stable EFR expression led to the activation of PAMP-triggered immune response in apple leaves upon treatment with supernatant of E.amylovora,as measured by the production of reactive oxygen species and the induction of known defense genes.The amount of tissue necrosis associated with E.amylovora infection was significantly reduced in the EFR transgenic rootstock compared to the wild-type.Our results show that the expression of EFR in apple rootstock may be a valuable biotechnology strategy to improve the resistance of apple to fire blight.

Breeding next generation tree fruits: technical and legal challenges

The new plant breeding technologies(NPBTs)have recently emerged as powerful tools in the context of‘green’biotechnologies.They have wide potential compared to classical genetic engineering and they are attracting the interest of politicians,stakeholders and citizens due to the revolutionary impact they may have on agriculture.Cisgenesis and genome editing potentially allow to obtain pathogen-resistant plants or plants with enhanced qualitative traits by introducing or disrupting specific genes in shorter times compared to traditional breeding programs and by means of minimal modifications in the plant genome.Grapevine,the most important fruit crop in the world from an economical point of view,is a peculiar case for NPBTs because of the load of cultural aspects,varietal traditions and consumer demands,which hinder the use of classical breeding techniques and,furthermore,the application of genetic engineering to wine grape cultivars.Here we explore the technical challenges which may hamper the application of cisgenesis and genome editing to this perennial plant,in particular focusing on the bottlenecks of the Agrobacterium-mediated gene transfer.In addition,strategies to eliminate undesired sequences from the genome and to choose proper target sites are discussed in light of peculiar features of this species.Furthermore is reported an update of the international legislative frameworks regulating NPBT products which shows conflicting positions and,in the case of the European Union,a prolonged lack of regulation.

Genome mapping of postzygotic hybrid necrosis in an interspecific pear population

Deleterious epistatic interactions in plant inter-and intraspecific hybrids can cause a phenomenon known as hybrid necrosis,characterized by a typical seedling phenotype whose main distinguishing features are dwarfism,tissue necrosis and in some cases lethality.Identification of the chromosome regions associated with this type of incompatibility is important not only to increase our understanding of the evolutionary diversification that led to speciation but also for breeding purposes.Development of molecular markers linked to the lethal genes will allow breeders to avoid incompatible inbred combinations that could affect the expression of important agronomic tratis co-segregating with these genes.Although hybrid necrosis has been reported in several plant taxa,including Rosaceae species,this phenomenon has not been described previously in pear.In the interspecific pear population resulting from a cross between PEAR3(Pyrus bretschneideri×Pyrus communis)and‘Moonglow’(P.communis),we observed two types of hybrid necrosis,expressed at different stages of plant development.Using a combination of previously mapped and newly developed genetic markers,we identified three chromosome regions associated with these two types of lethality,which were genetically independent.One type resulted from a negative epistatic interaction between a locus on linkage group 5(LG5)of PEAR3 and a locus on LG1 of‘Moonglow’,while the second type was due to a gene that maps to LG2 of PEAR3 and which either acts alone or more probably interacts with another gene of unknown location inherited from‘Moonglow’.

Transcriptional regulation of MdmiR285N microRNA in apple(Malus x domestica)and the heterologous plant system Arabidopsis thaliana

Malus x domestica microRNA MdmiR285N is a potential key regulator of plant immunity,as it has been predicted to target 35 RNA transcripts coding for different disease resistance proteins involved in plant defense to pathogens.In this study,the promoter region of MdmiR285N was isolated from the apple genome and analyzed in silico to detect potential regulatory regions controlling its transcription.A complex network of putative regulatory elements involved in plant growth and development,and in response to different hormones and stress conditions,was identified.Activity of theβ-Glucoronidase(GUS)reporter gene driven by the promoter of MdmiR285N was examined in transgenic apple,demonstrating that MdmiR285N was expressed during the vegetative growth phase.Similarly,in transgenic Arabidopsis thaliana,spatial and temporal patterns of GUS expression revealed that MdmiR285N was differentially regulated during seed germination,vegetative phase change,and reproductive development.To elucidate the role of MdmiR285N in plant immunity,MdmiR285N expression in wild-type apple plants and GUS activity in transgenic apple and Arabidopsis thaliana plants were monitored in response to Erwinia amylovora and Pseudomonas syringae pv.Tomato DC3000.A significant decrease of MdmiR285N levels and GUS expression was observed during host-pathogen infections.Overall,these data suggest that MdmiR285N is involved in the biotic stress response,plant growth,and reproductive development.