Abstract
Background"Candidatus Phytoplasma aurantifolia", is the causative agent of witches' broom disease in Mexican lime trees (Citrus aurantifolia L.), and is responsible for major losses of Mexican lime trees in Southern Iran and Oman. The pathogen is strictly biotrophic, and thus is completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. Therefore, we have applied a cDNA- amplified fragment length polymorphism (AFLP) approach to analyze gene expression in Mexican lime trees infected by "Ca. Phytoplasma aurantifolia".ResultsWe carried out cDNA-AFLP analysis on grafted infected Mexican lime trees of the susceptible cultivar at the representative symptoms stage. Selective amplifications with 43 primer combinations allowed the visualisation of 55 transcript-derived fragments that were expressed differentially between infected and non-infected leaves. We sequenced 51 fragments, 36 of which were identified as lime tree transcripts after homology searching. Of the 36 genes, 70.5% were down-regulated during infection and could be classified into various functional groups. We showed that Mexican lime tree genes that were homologous to known resistance genes tended to be repressed in response to infection. These included the genes for modifier of snc1 and autophagy protein 5. Furthermore, down-regulation of genes involved in metabolism, transcription, transport and cytoskeleton was observed, which included the genes for formin, importin β 3, transducin, L-asparaginase, glycerophosphoryl diester phosphodiesterase, and RNA polymerase β. In contrast, genes that encoded a proline-rich protein, ubiquitin-protein ligase, phosphatidyl glycerol specific phospholipase C-like, and serine/threonine-protein kinase were up-regulated during the infection.ConclusionThe present study identifies a number of candidate genes that might be involved in the interaction of Mexican lime trees with "Candidatus Phytoplasma aurantifolia". These results should help to elucidate the molecular basis of the infection process and to identify genes that could be targeted to increase plant resistance and inhibit the growth and reproduction of the pathogen.
Highlights
MethodsPlant material and inoculation Ten healthy 1-year-old Mexican lime trees grown in the greenhouse were used in this experiment
The present study identifies a number of candidate genes that might be involved in the interaction of Mexican lime trees with “Candidatus Phytoplasma aurantifolia”
Analysis with iPhyClassifier revealed that the virtual restriction fragment length polymorphism (RFLP) pattern that was derived from the phytoplasma 16 S rDNA fragment amplified from the diseased specimens was most similar to the reference pattern of the 16Sr group II, subgroup B phytoplasma (GenBank accession: U15442), with a pattern similarity coefficient of 0.99
Summary
Plant material and inoculation Ten healthy 1-year-old Mexican lime trees grown in the greenhouse were used in this experiment. Specimens from Mexican lime trees infected with witches’ broom were grafted to healthy trees, and specimens from healthy Mexican lime trees were grafted to other healthy trees. The grafted plants were covered for 1 month with plastic bags to increase humidity and were arranged randomly on the greenhouse bench. They were kept under natural light conditions at a temperature of 25-28°C. The branches infected with witches’ broom were sampled 20 weeks after inoculation and used for RNA extraction. RNA was extracted from non-grafted healthy plant leaves that has been grown under similar conditions
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