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Apple proliferation (AP) is one of the most serious diseases of apple trees in Europe. It is caused by a phytoplasma, Candidatus Phytoplasma mali. The goal of the present study was to analyze transcriptional profiles of Malus pumila during infection by Ca. P. mali using cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique in order to gain insight into molecular and physiological changes in diseased plants. We used a rootstock of apple (MM106) susceptible to Ca. P. mali to maximise the range of the potential host responses, and two strains (AP and AT) of the pathogen. Gene expression comparisons were studied in 3 categories of plant materials: healthy sample versus infected samples, symptomatic versus non-symptomatic sample, and AP-infected sample versus AT-infected sample. Forty-five genes whose steady-state levels of expression significantly changed in response to phytoplasma infection were isolated and identified. Of 45 partial cDNA sequences, twenty-seven showed similarity to international DNA or protein data bases. Of these, 18 were previously characterized in plants (the rest was related to unknown or hypothetical proteins). Eighteen out of 45 did not show any similarity with sequences in data bases, and so may be present novel genes. The majority of fragments were differently expressed between healthy sample and infected samples (fewer differences between symptomatic and non-symptomatic samples, or between the samples infected by different strains of phytoplasma). Quantitative Real-time RT-PCR (qRT-PCR) was used to confirm differential expression of sequences isolated by cDNA-AFLP. We chose the most stable reference housekeeping genes (GAPDH and actin) for normalisation of our data. The gene expression ratios were calculated by means of ΔΔCt method. Consequently, the second methodology (qRT-PCR) showed the similar profile expression as primary elucidation technique (cDNA-AFLP) for 11 known genes (between 18) and 13 unknown, hypothetical or novel genes (between 27). Changes in gene expression involved a wide spectrum of biological functions, including processes of metabolism, cell defence, senescence, photosynthesis, transport, transcription, signal transduction and protein synthesis. The possible effect of phytoplasma infection on these processes and their relationships with disease development, symptom appearance and probably plant defence system is discussed. A model is proposed to explain the mode of action of the Ca. P. mali in its host plant, apple tree. This is the first study of global gene profiling in plants in response to phytoplasma infections using cDNA-AFLP. |
