Chasing consistency: an update of the TCP gene family of Malus × domestica

The 52 members of the Teosinte-Branched 1/Cycloidea/Proliferating Cell Factors (TCP) Transcription Factor gene family in Malus × domestica (M. × domestica) were identified in 2014 on the first genome assembly, which was released in 2010. In 2017, a higher quality genome assembly for apple was released and is now considered to be the reference genome. Moreover, as in several other species, the identified TCP genes were named based on the relative position of the genes on the chromosomes. The present work consists of an update of the TCP gene family based on the latest genome assembly of M. × domestica. Compared to the previous classification, the number of TCP genes decreased from 52 to 40 as a result of the addition of three sequences and the deduction of 15. An analysis of the intragenic identity led to the identification of 15 pairs of orthologs, shedding light on the forces that shaped the evolution of this gene family. Furthermore, a revised nomenclature system is proposed that is based both on the intragenic identity and the homology with Arabidopsis thaliana (A. thaliana) TCPs in an effort to set a common standard for the TCP classification that will facilitate any future interspecific analysi

Expression of a bacterial effector, harpin N, causes increased resistance to fire blight in Pyrus communis

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. In order to create novel mechanisms for fire blight resistance in pear, we have generated transgenic pears expressing the elicitor harpin Nea from Erwinia amylovora under the control of the constitutive promoter CaMV35S. The transient expression of hrpN Ea in pear cells did not provoke any apparent damage. Therefore, stable constitutive expression of hrpN Ea was studied in seventeen transgenic clones of the very susceptible cultivar "Passe Crassane.” Most transgenic clones displayed significant reduction of susceptibility to fire blight in vitro when inoculated by E. amylovora, which was positively correlated to their degree of expression of the transgene hrpN Ea . These results indicate that ectopic expression of a bacterial elicitor such as harpin Nea is a promising way to improve pear resistance to fire bligh

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 the \u3b2-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

Activation of the Pathogen-Inducible Gst1 Promoter of Potato after Elicitation by Venturia inaequalis and Erwinia amylovora in Transgenic Apple ( Malus × Domestica )

Rather than using a constitutive promoter to drive transgenes for resistance against fungal and bacterial diseases in genetic engineering of apple (Malus × domestica) cultivars, a promoter induced only after infection was preferred. The ability of the Pgst1 promoter from potato (Solanum tuberosum L.) to drive expression of the gusA reporter gene was determined in two genotypes of apple: the fruit cultivar Royal Gala and the M.26 rootstock. β-glucuronidase activity in the transgenic lines grown in a growth chamber was determined quantitatively using fluorometric assays and compared to the activity in Cauliflower Mosaic Virus (CaMV) 35S promoter-driven transgenic lines. In both apple genotypes, the Pgst1 promoter exhibited a low level of expression after bacterial and fungal inoculation compared to the level obtained with the PCaMV35S promoter (15% and 8% respectively). The Pgst1 promoter was systematically activated in apple at the site of infection with a fungal pathogen. It was also activated after treatment with salicylic acid, but not after wounding. Taken together, these data show that, although the Pgst1 promoter is less active than the PCaMV35S promoter in apple, its pathogen responsiveness could be useful in driving the expression of transgenes to promote bacterial and fungal disease resistanc

Mfu16 is an unstable fire blight resistance QTL on linkage group 16 of Malus fusca MAL0045

A strong fire blight resistance QTL (Mfu10) was previously detected on linkage group 10 (LG10) of Malus fusca accession MAL0045, using several strains of the causative bacterium, Erwinia amylovora. As no strain capable of breaking the resistance of MAL0045 has been found, we hypothesized that another locus contributes to its fire blight resistance. However, none was detected with strains previously tested on the progeny. Here, an avrRpt2EA mutant strain (Ea1038) with the chromosomal S-allele deleted and complemented with the less aggressive C-allele, was used to phenotype MAL0045 × ‘Idared’ progeny. We performed phenotype-genotype analyses using the first genetic map of MAL0045, which is scarcely dense, and a recently constructed saturated map. As expected, Mfu10 was detected on LG10 with Ea1038, as was previously with other strains. Interestingly, a QTL with a logarithm of odds (LOD) thresholds of 5.5 and 2.9, significant at the genome-wide and chromosome levels, respectively, was detected with Ea1038 on LG16 (Mfu16) in a subset of 76 individuals, but only using the saturated map. Progenies carrying both Mfu10 and Mfu16 were significantly more resistant than progenies carrying only Mfu10. However, the LOD of Mfu16 diminished to 2.6 in a larger subset of individuals. We hypothesize that Mfu16 is present in the genome of MAL0045 albeit unstable in the progeny

Transcriptomic and volatilomic profiles reveal Neofabraea vagabunda infection-induced changes in susceptible and resistant apples during storage

Bull’s eye rot is one of the most severe diseases that may affect apple fruit during post-harvest storage. It is caused by the fungus Neofabraea vagabunda, and the mechanism by which the pathogen infects the fruits is only partially understood. In particular, very little is known about the molecular mechanisms regulating the interaction between the pathogen and the host during symptoms development. Despite different apple cultivars show divergent levels of resistance to the pathogen, the genetic basis of these responses is still unknown. In order to better understand the molecular mechanisms occurring in the apple fruit during N. vagabunda infection, a large-scale transcriptome study by RNA-Seq analysis was performed, comparing fruits of the sensitive ‘Roho’ cultivar and the resistant cultivar ‘Ariane’ after artificial infection with N. vagabunda and a storage period of four months. Transcriptomic analyses revealed the regulation of several classes of genes during this period, some of which may be involved in apple-pathogen interaction, such as superoxide dismutases and heat shock proteins (HSPs), ethylene-responsive transcription factors (ERFs), carboxylesterases and NAC transcription factors gene families. Moreover, a volatile analysis was performed, revealing differences in the volatile profile between the resistant and the susceptible cultivar that may help to elucidate the resistance mechanism. RNA-Seq data highlighted several classes of pathogen-related genes, such as genes coding for enzymes involved in cell wall disruption and in reactive oxygen species (ROS) homeostasis, being differentially regulated between resistant and susceptible fruits and between diseased and healthy fruits of the same cultivar, indicating that apples are capable of perceiving and triggering a molecular response to N. vagabunda infection. Some volatiles, as ethanol and methanol, but also furan and formaldehyde, might be potential markers for N. vagabunda infection; others, such as hexenal and methyl acetate, were found to be putatively involved in regulating apple-fungi interactio