Characterization and in vitro expression patterns of an exopolygalacturonase encoding gene from Fusarium oxysporum f.sp. radicis lycopersici.

AIMS: In this work, we report the isolation, characterization and expression pattern in in vitro cultures of an EXOPG encoding gene (pgx2), a novel EXOPG encoding gene of Fusarium oxysporum f.sp. radicis lycopersici, responsible for foot crown and root rot disease in tomato plants. The gene was compared with other fungal polygalacturonases (PGs) previously reported. METHODS AND RESULTS: Partial sequences of the purified EXOPG native protein were used to design primers that amplified a genomic fragment by PCR. The amplified genomic fragment was used as a probe to screen a genomic library. One isolated clone was analysed. The complete genomic, cDNA and the deduced amino acid sequences were compared with other fungal EXOPGs and ENDOPGs. Regulation of pgx2 expression was analysed by Northern blot in in vitro cultures supplemented with different carbon sources. CONCLUSIONS: Pgx2 was present as single copy in the haploid genome of several Fusarium species. PGX2 showed the conserved amino acid motifs typical of PGs and those reported for fungal EXOPGs. Pgx2 was regulated at transcriptional level showing similar expression pattern to other EXOPG encoding gene (pgx1) when the fungus was cultured on different carbon sources suggesting a coordinate expression of both genes. This similarity would be supported by the presence of common putative regulatory motifs in the upstream regions of both genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the analysis of a novel EXOPG gene of the tomato pathogen F. oxysporum f.sp. radicis lycopersici, a contribution to the understanding of the role of cell-wall-degrading enzymes produced by fungi during pathogenesis.

The course of pectin degradation by polygalacturonases from Fusarium oxysporum f. sp. radicis lycopersici.

The production of polygalacturonases from Fusarium oxysporum f. sp. radicis lycopersici (FORL) occurred sequentially, and endo- and exo-polygalacturonases (PG) predominated at different times during growth. At first FORL produced a PG which did not release monomers from pectins and was unable to hydrolyse dimers and trimers. Later a complex was produced in which exo-PG seemed to be the predominant activity; the monomer was released rapidly and this activity was able to degrade dimers and trimers.

Control of polygalacturonase synthesis in Fusarium oxysporum f.sp. radicis lycopersici.

Genetic control of polygalacturonase (PG) activity from Fusarium oxysporum f.sp. radicis lycopersici was analyzed on pectin and glucose cultures. One exopolygalacturonase from F. oxysporum f.sp. radicis lycopersici was strongly induced, in stationary culture, when the fungus was grown on apple pectin, while on glucose no extracellular PG activity could be detected. Although SDS-PAGE detected the presence of a putative PG band (66 kDa) in both conditions, specific antibodies obtained against the purified PG only detected it in PG-inducing conditions, that is to say, when apple pectin was used as the carbon source. Northern blot analysis of RNA of two isolates of F. oxysporum f.sp. radicis lycopersici (r6 and r2) confirmed that this regulation of PG synthesis was exerted at the transcriptional level. Only one single mRNA species of around 1400 nucleotides was detected on the cultures containing pectin and was absent in glucose-grown cultures. Southern blot analysis of genomic DNA indicated that pg gene seems to be present in a single copy in the genomes of F. oxysporum f.sp. radicis lycopersici r6 and r2 and Fusarium oxysporum f.sp. lycopersici, showing similar hybridization patterns in all species. The partial sequence of this pg gene from F. oxysporum f.sp. radicis lycopersici r6, which is also reported, showed high similarity to diverse PGs already reported. Exopolygalacturonase of F. oxysporum f.sp. radicis lycopersici r6 is heavily glycosylated; its deglycosylated form had a molecular mass of 50 kDa.