Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum.

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of wheat worldwide, resulting in yield losses and mycotoxin contamination. The molecular mechanisms regulating Fusarium penetration and infection are poorly understood. Beside mycotoxin production, cell wall degradation may play a role in the development of FHB. Many fungal pathogens secrete polygalacturonases (PGs) during the early stages of infection, and plants have evolved polygalacturonase-inhibiting proteins (PGIPs) to restrict pectin degradation during fungal infection. To investigate the role of plant PGIPs in restricting the development of FHB symptoms, we first used Arabidopsis thaliana, whose genome encodes two PGIPs (AtPGIP1 and AtPGIP2). Arabidopsis transgenic plants expressing either of these PGIPs under control of the CaMV 35S promoter accumulate inhibitory activity against F. graminearum PG in their inflorescences, and show increased resistance to FHB. Second, transgenic wheat plants expressing the bean PvPGIP2 in their flowers also had a significant reduction of symptoms when infected with F. graminearum. Our data suggest that PGs likely play a role in F. graminearum infection of floral tissues, and that PGIPs incorporated into wheat may be important for increased resistance to FHB.

A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition.

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.

The characterization of the soybean polygalacturonase-inhibiting proteins (Pgip) gene family reveals that a single member is responsible for the activity detected in soybean tissues.

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit fungal endopolygalacturonases (PGs). They are encoded by multigene families whose members show functional redundancy and subfunctionalization for recognition of fungal PGs. In order to expand the information on the structure and functional features of legume PGIP, we have isolated and characterized four members of the soybean Pgip gene family and determined the properties of the encoded protein products. Sequence analysis showed that these genes form two clusters: one cluster of about 5 kbp containing Gmpgip1 and Gmpgip2, and the other containing Gmpgip3 and Gmpgip4 within a 60 kb fragment of a separate BAC clone. Sequence diversification of the four members resides mainly in the xxLxLxx region that includes residues forming the beta-sheet B1. When compared with other legume Pgip genes, Gmpgip3 groups with the bean genes Pvpgip1 and Pvpgip2, suggesting that these genes are closer to the ancestral gene. At the protein level, only GmPGIP3 shows the capability to inhibit fungal PGs. The spectrum of inhibition of GmPGIP3 against eight different fungal PGs mirrors that of the PGIP purified from soybean tissues and is similar to that of the bean PvPGIP2, one of the most efficient inhibitors so far characterized. We also report that the four Gmpgip genes are differentially regulated after wounding or during infection with the fungal pathogen Sclerotinia sclerotiorum. Following fungal infection Gmpgip3 is up regulated promptly, while Gmpgip2 is delayed.

Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein.

A polygalacturonase-inhibiting protein (PGIP) was detected in soybean (Glycine max (L.) Merr.) seedlings. The protein was purified from germinating seeds and appeared to consist of at least three components with very close molecular weights (between 37 and 40 kDa) but each showing a unique N-terminal sequence. Primers specific for N-terminal and C-terminal nucleotide sequences of field bean (Phaseolus vulgaris L.) PGIP were used in a polymerase chain reaction (PCR) on soybean DNA, and only one amplification band was obtained. The amplified product was cloned and one of the PCR clones was sequenced. The nucleotide sequence comprises 942 bp with a single open reading frame which encodes a polypeptide of 313 amino-acid residues with a predicted molecular weight of 33984 Daltons and an isoelectric point of 8.21. Analysis of genome organization showed a single gene copy of PGIP with few related sequences, and wounding of soybean hypocotyls showed a strong induction of expression of the PGIP gene. The PGIP showed different activities toward three purified fungal endo-polygalacturonases (endo-PGs) (two endo-PGs from Sclerotinia sclerotiorum and one endo-PG from Aspergillus niger). A possible involvement of soybean PGIP in plant defence against fungal pathogens is discussed.

Expression and localization of polygalacturonase during the outgrowth of lateral roots in Allium porrum L.

The presence of polygalacturonase and its correlation with the formation of lateral roots in leek (Allium porrum L.) seedlings have been investigated. During root growth, a steady increase in polygalacturonase activity was associated with that of the lateral root primordia. Fractionation of root extract by fast protein liquid chromatography resolved at least two polygalacturonase isoforms. One of the isoforms, a 75-kdalton protein, strongly reacted on Western blots probed with a polyclonal antibody raised against tomato polygalacturonase. It also reacted with both polyclonal and monoclonal antisera raised against Fusarium moniliforme polygalacturonase. In situ localization with these three antibodies showed that polygalacturonase was present over the meristems of lateral root primordia. Antibodies against pectins (Knox et al. 1990, Planta 181, 512-521) detected large amounts of pectic material filling the area between the apex of the primordium and the mother root tissues. We suggest that a polygalacturonase plays an important role in leek root morphogenesis, particularly during lateral root outgrowth.