Investigating the molecular targets of Apple Proliferation Phytoplasma effector SAP11CaPM in apple

Apple Proliferation (AP) is a phytoplasma-related associated with 'Candidatus Phytoplasma mali' ('Ca. P. mali'). Trees affected by the disease display several symptoms associated with plant shape and development, ultimately leading to the generation of non-marketable fruits. The disease is widespread in many apple cultivated areas of Europe, where outbreaks in recent years caused huge economic losses. There is no cure or treatment for phytoplasmoses but to control the vector insects, the uprooting of affected trees and the use of healthy propagation material. In the last years, extensive research led to the identification of AP-tolerant experimental rootstock, but the molecular basis of this inheritable trait is unknown. Phytoplasmas secrete pathogenic proteins, called effectors, that affect the host physiology and determine a fitness advantage for the bacteria. Among these, the best characterized in 'Ca. P. mali' is SAP11CaPM, which was observed to target and deactivate two M. × domestica transcription factors of the TCP family, i.e., MdTCP4a and MdTCP13a. This work aims to investigate the molecular targets of SAP11CaPM to shed light on the molecular basis of disease development and the phenomenon of tolerance. The second chapter consists of a revision of the MdTCP gene family set by performing a re-identification on the latest high-quality M. × domestica genome assembly. Of the 52 MdTCP sequences previously described, 15 were discarded because of redundancy with other MdTCPs (six) or the absence of a complete open reading frame (nine). Of the 37 remaining MdTCP sequences, 30 were observed to be included in the list of functionally annotated genes automatically computed. Furthermore, three novel MdTCP sequences have been identified, for a total of 40 sequences constituting the revised set of the MdTCP gene family. Analysis of the intergenic identity, combined with the synteny analysis previously performed on the whole M. × domestica genome, allowed the identification of 15 pairs of genes that likely originated through a recent whole-genome duplication event. Finally, the 40 MdTCP sequences identified were named according to the homology with A. thaliana TCP. In chapter 3, the sequences of the two SAP11CaPM-target genes have been analyzed in susceptible and tolerant genotypes to screen for potential differences that could explain the tolerant behaviour displayed by some plants. Sequence analyses led to the identification of two rare amino acid substitutions in MdTCP4a, of which one, in particular, was not found in any of the TCP sequences published to date. The interaction between the bacterial effector and the protein alternatives from susceptible and tolerant genotypes was measured both in a heterologous system (Y2H) and in planta (BiFC). The interaction assays showed no difference in the interaction strength between the different alternatives, suggesting that this mechanism cannot explain the tolerant phenotype. Nonetheless, a good degree of correlation between the presence of the two amino acid substitutions and tolerance to AP was found, indicating a possible role of these sequences as genetic markers of tolerance. Finally, chapter 4 describes the generation and characterization of M. × domestica transgenic plants overexpressing the two MdTCP genes via At-mediated transformation. While plants overexpressing MdTCP13a died shortly after the regeneration, suggesting that overexpression of this gene can hinder the plant development, several independent MdTCP4a-overexpressing transgenic lines could be generated. Transgenic lines kept in vitro showed a low level of overexpression and no differences in phenotype compared to non-transformed plants. Following the soil acclimatization, the transgenic plants demonstrated an increase in the overexpression level and displayed some peculiar phenotype characteristics, such as smaller and crinkled leaves, loss of apical dominance, and generation of more shoots than non-transformed. In vitro transgenic plants were infected with 'Ca. P. mali' using the micrografting technique and were screened for the presence of phytoplasma with a qPCR assay. Results show that two out of three transgenic lines tested display a phytoplasma concentration significantly lower than non-transformed, which is commonly associated with less severity or complete absence of symptoms. Furthermore, the transcriptomes of three soil acclimatized lines have been analyzed via RNAseq, and preliminary analyses show an up-regulation of several genes associated with plant development and abiotic stress responses.