Citrus variegated chlorosis: Development of transgenic resistance and molecular studies of pathogenesis

Xylella fastidiosa is a xylem-inhabiting pathogenic that is spread by insect vectors and which causes severe plant diseases. Its localization in the xylem poses a challenge for the development of transgenic resistance against this pathogen. Mature xylem vessels are composed of dead cells that lack intrinsic metabolic activity, but are surrounded by living xylem parenchyma that remain active throughout the life of the plant. Xylem parenchyma shows high activity of the enzyme phenylalanine ammonia-lyase (PAL), which is required for the synthesis of phenylpropanoid precursors of the lignin that reinforces the walls of xylem vessels. The high level of expression and tissue specificity of the PAL gene make the PAL promoter a potentially useful candidate for development of transgenic plants that produce antimicrobial peptides in xylem tissues. A PAL promoter was cloned from sweet orange through inverse-PCR and demonstrated to express GUS preferentially in the xylem of transgenic tobacco. A gene for the antibacterial peptide cecropin MB39, preceded by a signal peptide for export, was synthesized by PCR. Tobacco was demonstrated to be colonized by X. fastidiosa, and used as a model to study the effect of cecropin gene expression against infection by this pathogen. Tobacco plants were transformed with a construct containing the cecropin gene under control of the sweet orange PAL promoter and transgene expression was demonstrated.; Genetic comparison of CVC and PD strains of X. fastidiosa was performed using a suppression subtractive hybridization procedure. Sequencing of 18 clones revealed that the PD strain lacks a 51 kb plasmid present in the CVC strain, and that bacteriophage-related genes are responsible for some of the differences between the strains.