Cloning, Expression, and Role in Pathogenicity of pg1 Encoding the Major Extracellular Endopolygalacturonase of the Vascular Wilt Pathogen Fusarium oxysporum

Abstract

pg1 encoding the major in vitro extracellular endopolygalacturonase of the tomato vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici was cloned and sequenced. The deduced mature protein had a calculated molecular mass of 35.5 kDa and a pI of 6.2, and showed significant similarity with other fungal endoPGs. pg1 mRNA was induced in vitro by citrus pectin, tomato vascular tissue, 0.1% D-galacturonic acid, and polygalacturonic acid, and repressed by 1% D-galacturonic acid and 1% glucose. Reverse transcription-polymerase chain reaction revealed pg1 expression in roots and lower stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Three naturally occurring F. oxysporum f. sp. melonis isolates deficient in PG1 were transformed with the cloned gene. The PG1 enzyme secreted by the transformants had the same molecular mass, pI, and glycosylation pattern as those of the donor isolate. Polygalacturonase activity in cultures of transformants grown in vitro on citrus pectin and on melon plants, but not on glucose, increased 10- to 20-fold, compared with the PG1-deficient wild-type isolate, whereas mycelial dry weight increased two- to three-fold. Transformants exhibited the same degree of virulence toward susceptible muskmelon cultivars as the wild-type isolate and were avirulent on a resistant cultivar.

Role of chitin synthase genes in Fusarium oxysporum

“…Two-week-old plants were inoculated by immersing their roots in a microconidial suspension of the wild-type strain, or the disruptants Dchs1, Dchs2 and Dchs7. Plants were scored for vascular wilt symptoms at different time intervals (Di Pietro & Roncero, 1998). The development of the disease is shown in Fig.…”

“…For extraction of genomic DNA, mycelium was obtained from cultures grown in potato dextrose [glucose] broth (PDB, Difco) on a rotary shaker at 170 r.p.m. and 28 uC as described previously (Di Pietro & Roncero, 1998). For phenotypic analysis of colony growth inhibition or hydrophobic characteristics, microconidia were collected from PDB, washed in sterile water, counted and transferred to synthetic medium (SM) plates (Di Pietro & Roncero, 1998) supplemented or not with the following metabolites at the concentrations indicated: sorbitol, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), caffeine, a-tomatine, Congo red and hydrogen peroxide (all from Sigma).…”

“…3(a). the chs coding regions interrupted with the Hyg R cassette were used for transformation of F. oxysporum 4287 protoplasts to hygromycin resistance according to a protocol described previously (Di Pietro & Roncero, 1998). Briefly, microconidia were germinated for 14 h in SM before being submitted to protoplasting (García-Maceira et al, 2000).…”

“…Infection of tomato plants was performed as reported previously (Di Pietro & Roncero, 1998). Briefly, tomato seedlings of cv.…”

“…Plants immersed in sterile water were used as controls. For statistical analyses, the severity of disease symptoms was recorded from 1 week after the inoculation every 2 days until day 24 post-infection according to a scale ranging from 1 (healthy plant) to 5 (dead plant) (Di Pietro & Roncero, 1998). Fifteen plants were used for each treatment.…”