Shotgun Proteomics Characterizes the Bacterial Infection of Candidatus Phytoplasma aurantifolia in Mexican Lime Trees

Witches’-broom disease, caused by the bacterium Candidatus Phytoplasma aurantifolia, has the potential to cause devastating loss of citrus trees. The disease is prevalent in Asia and North Africa, and it is estimated that 30% of lime trees have been destroyed by this bacterium in Iran.¹ To investigate the interactions occurring during infection with Witches’-broom disease, the Salekdeh group performed a shotgun proteomic analysis of a Mexican lime tree (Citrus aurantifolia L.) infected with Candidatus Phytoplasma aurantifolia.²

For this investigation, 10 healthy Mexican lime tree seedlings were obtained and grown in a greenhouse. Specimens infected with Candidatus Phytoplasma aurantifolia were grafted into five seedlings. Healthy specimens of C. aurantifolia were grafted onto the healthy trees. In approximately 20 weeks, trees with Candidatus Phytoplasma aurantifolia showed typical symptoms of the disease, while the remaining five seedlings were healthy.

Proteins from infected and diseased trees were extracted and separated via SDS-PAGE. Proteins were digested, and tryptic peptides were separated by reverse-phase chromatography. Analysis was carried out using an LTQ-XL ion-trap mass spectrometer (Thermo Scientific). Peptides from each fraction were injected onto the C18 column using a Surveyor Autosampler (Thermo Scientific). Mass spectra were analyzed using Xcalibur software, version 2.06 (Thermo Scientific).

False discovery rates were calculated as being less than 1%, with reversed database hits and contaminants excluded with p values < 0.05 as calculated via t-tests. Furthermore, only proteins that were present in all replicates for at least one condition (healthy or diseased) were included in the data set.

Mass spectra was searched using an in-house transcriptome database of the C. aurantifolia. Results of the analysis identified 990 nonredundant proteins. A total of 542 of the 990 proteins identified did not differ significantly between healthy and infected plants; however, the abundance of 448 proteins changed significantly in response to the pathogen. Of these 448 proteins, 274 were increased, and 174 were decreased in abundance in response to biotic stress. A total of 360 proteins were grouped by function. These proteins were involved with protein, lipid, and carbohydrate metabolism; growth and development; signal transduction; and cell cycle and cell wall organization. In addition, proteins were identified as generators of precursor metabolites and as being involved in photosynthesis.

In addition, 144 of the differentially accumulated proteins could be categorized as being related to biotic stress. The Salekdeh group hypothesized that the down-accumulation of nearly 61% of the differentially accumulated proteins was due to the exploitation of cellular resources and/or the suppression of defense responses as a result of the host infection.

This work brings new insights into the reprogramming of the primary and secondary metabolic pathways involved in the host infection as well as the activation of proteins responsible for defense mechanisms. It may also lead to future prevention and treatment of this infectious pathogen.

References

1. Mardi, M., et al. (2011) ‘Witches’ broom disease of Mexican lime trees: disaster to be addressed before it will be too late‘, Bulletin of Insectology, 64 (Supplement), (pp. S205-S206)

2. Monavarfeshani, A., et al. (2013) ‘Shotgun proteomic analysis of the Mexican lime tree infected with “CandidatusPhytoplasma aurantifolia‘, Journal of Proteome Research, 12 (2), (pp. 785-795)