Respuestas orales

The data obtained from the gene expression analysis were in several cases affected by variability. The cDNA-AFLP data and the real-time PCR data were often not in agreement. Although the gene expression pattern were analysed in stably infected in vitro-grown plants, this variation can be attributed in part to the different plant material used for the two analyses. As discussed above, cDNA-AFLP data have technical limits and need confirmation by an independent method. In this respect, qRT-PCR yields data that are more reliable and, therefore, the interpretation of the gene expression results was mainly based on the real-time PCR data. Although the in vitro system has been proven to be a powerful tool for the study of the resistance to AP in apple plants, the genes individuated as differentially expressed had to be confirmed in plants grown in conditions similar to those present in the orchards. Therefore, alternatively to further qRT- PCR analysis on the in vitro plants, ex vitro plants were used to assess the expression of the analysed genes. The interpretation of the data is also limited by the high number of genes for which no function could be assigned (genes 3, 14, 21, 22, 28, 30, 31, 32 ). E.g., the differential expression of gene 22 indicate a possible involvement in the resistance phenotype but no hypothesis of the resistance mechanism can be deduced because of its unknown function.

The determination of the entire genome sequence of „Ca. P. mali‟ (Kube et al., 2008) revealed that the phytoplasma lacks a wide range of metabolic pathways and is dependent on a multitude of plant metabolites. Biochemical measurements of the compounds produced or degraded during the infection showed a broad spectrum of metabolisms involved in the plant- phytoplasma interaction (Kartte and Seemüller, 1991; Lepka et al., 1999; Choi et al., 2004). Consequently, genes involved in various metabolic pathways have been found to be differentially regulated after phytoplasma infection by other studies (Carginale et al., 2004; Aldaghi, 2009). This further complicated the individuation of genes directly involved in the resistance response against

The gene expression data obtained in this thesis demonstrate that „Ca. P. mali‟ induces a general stress response in apple plants. Based on the genes which had homology to known sequences genes associated with general plant defence, energy transport/oxidative stress response, protein metabolism and cellular growth were identified.

Genes 5, 24, 27 and 29 are related to the general plant defence induced upon pathogen attack or abiotic stress. It is not clear if these genes are activated by a general stress due to the presence of the phytoplasma (blocking of sieve tubes, depletion of metabolites etc.) or because of the identification of molecules that reveal its presence in the host. Although the genes 5 (chitinase I) and 29 (Hsp70) are homologous to proteins known to be involved in the general plant response, the genes 24 (AKINgamma) and 27 (BON1/CPN family) are good candidates for further investigations. AKIN proteins are involved in the assembly of the SnRK1 kinase complex which plays a role in plant-pathogen interaction (Hao et al., 2003; Gissot et al., 2006) and as cellular energy sensor regulating the resources allocation between plant organs (Schwachtje et al., 2006; Lee et al., 2009). Therefore, the gene 24 together with the gene 27 which has a role in the regulation of the disease resistance gene SNC1 merit further investigation as candidates for a specific answer in the resistant genotypes.

Genes 6, 9, 25 and 33 show homology to genes involved in energy transfer and oxidative metabolisms. Although differential expression was observed only in some genotypes this group could be interesting for further studies. Physiological studies in AP-diseased apple plants showed that the presence of the phytoplasma has a negative influx on the photosystems and in the discharge of the electron transfer chains (Bertamini et al., 2002; Bertamini et al., 2003). A resulting effect is the generation of reactive oxygen species (ROS) with subsequent production of hydrogen peroxide (H2O2). As mentioned above for the gene 33, glycolate oxidase can be involved in

defensive mechanisms with generation of H2O2 (Corpas et al., 2001; del Rio et al., 2003; Palma et

al., 2009). H2O2 is involved in pathogen response (Baker and Orlandi, 1995) and activates a series

of responses like the sterol pathway of which squalene monoxygenase (gene 25 and 35) is a key enzyme (Pose et al., 2009). Because of the toxicity of H2O2 its effect is modulated in defence

responses by ROS scavengers including metallothioneins (gene 9) which expression was shown to affect pathogen susceptibility to both bacterial blight and blast infection in rice (Oryza sativa) (Wong et al., 2004). Interestingly, this gene in ex vitro plants of the resistant genotypes shows down-regulation in roots and up-regulation in leaf tissues.

The product of the gene 9 could be also associated, as well as the gene 11, to mechanisms related with protein degradation and aminoacid metabolisms. The phytoplasmas could specifically stimulate these pathways to render available basic molecules for their metabolism (Carginale et al., 2004) or could imbalance the phloematic transport of nutrients unspecifically affecting the plant metabolism (Seemüller et al., 1984; Lepka et al., 1999).

The last functional group, to which the gene 7 and 15 belongs, was associated to genes that may be involved in the regulation of the plant development. Both showed differential expression in

ex vitro plants not supported by the results in in vitro tissues. Further analyses are necessary to

better characterise their expression behaviour. These genes could give insights on how the phytoplasma acts inducing dramatic changes in plant phenotype. Very little is known on this aspect of the AP-infection where the development of the symptoms suggest a strong modification of the signals and the pathways that regulate the cell growth but less is known on which pathways are involved.

Two genes (gene 14, 32) with unknown function showed a „Ca. P. mali‟ strain-specific differential expression. This indicates that the plant specifically reacts to the phytoplasma strain. Differences in virulence among „Ca. P. mali‟ strains (Seemüller and Schneider, 2007), as well as differences in phytoplasma concentration in the studied in vitro plants (Bisognin et al., 2008a) support this strain-specific pathogen-plant interaction. This result further increases the complexity of the interaction between „Ca. P. mali‟ and the plant.