Analysis of the intercellular fluid proteome of Xylella fastidiosa -
infected citrus leavesIn order to understand the mechanisms regulating the Xylellafastidiosacitrus (Citrus sinensis var. Pêra) interaction,
proteins withdifferential
accumulation in the apoplast of Xf-infected citrus leaveswith or without disease
symptoms as
compared to non-infected leaves,were identified and characterized using 2D-PAGE and mass spectrometry(MALDI-ToF). The intercellular fluid (IF) was extracted by infiltrationand centrifugation from field grown leaves segments. The presence of Xfin the leaves was determined by PCR-amplification of specific DNAfragments. SDS-PAGE was performed in a discontinuous system using equalamounts of IF proteins from non-infected and infected leaves with orwithout CVC symptoms collected from field grow plants at diferrentlocations.
protein profiles were compared using discriminant analyses,based on the relative abundance of each protein. The results indicatedthat the IF proteome of plants from different localities are distinct,independent of the presence of Xf and/or CVC symptoms. Also,independent of the local where the samples were collected, the IFproteome of non-infected leaves and symptomatic infected leaves weredistinct. Using 2D-PAGE it was possible to identify proteins withdifferential accumulation in the IF of symptomatic and asymptomatic Xfinfected citrus leaves, as compared to noninfected controls. The mustabundant proteins in the IF showing differential accumulation haveapparent molecular mass of 41kDa and pI 4,2-5. The accumulation in theIF asymptomatic Xf infected citrus leaves were 89-129% higher thannon-infected controls. Their N-terminal position and the mass spectraof their peptides, after trypsin digestion, are mostly identical,indicating that the are isoforms of the same proteins familiy.N-terminal sequences of the proteins and their internal peptides showedno homology to known genes/proteins in public database, suggesting thatthese proteins are apoplastic citros specifid proteins, and that theymay have a important roles in regulating citrus-Xf interactions.
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