The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed beta-strand/beta-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability.

Two members of the pgip gene family (pgip-1 and pgip-2) of Phaseolus vulgaris L. were expressed separately in Nicotiana benthamiana and the ligand specificity of their products was analysed by surface plasmon resonance (SPR). Polygalacturonase-inhibiting protein-1 (PGIP-1) was unable to interact with PG from Fusarium moniliforme and interacted with PG from Aspergillus niger; PGIP-2 interacted with both PGs. Only eight amino acid variations distinguish the two proteins: five of them are confined within the beta-sheet/beta-turn structure and two of them are contiguous to this region. By site-directed mutagenesis, each of the variant amino acids of PGIP-2 was replaced with the corresponding amino acid of PGIP-1, in a loss-of-function approach. The mutated PGIP-2s were expressed individually in N.benthamiana, purified and subjected to SPR analysis. Each single mutation caused a decrease in affinity for PG from F.moniliforme; residue Q253 made a major contribution, and its replacement with a lysine led to a dramatic reduction in the binding energy of the complex. Conversely, in a gain-of-function approach, amino acid K253 of PGIP-1 was mutated into the corresponding amino acid of PGIP-2, a glutamine. With this single mutation, PGIP-1 acquired the ability to interact with F.moniliforme PG.

Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris.

The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.

Regulation of a carotenoid biosynthesis gene promoter during plant development.

Carotenoids are terpenoid pigments which are accumulated in the chloroplasts of leaves and in the chromoplasts of many flowers and fruits. Phytoene desaturase (Pds), the second dedicated enzyme in carotenoid biosynthesis, is encoded in tomato by a single copy gene. A 2 kb fragment from the tomato Pds gene, comprising 1.5 kb from the promoter and 0.5 kb from the 5' non-translated region, is able to drive developmentally regulated expression of the GUS reporter gene in transgenic tomato and tobacco plants. In tomato, high levels of Pds/GUS expression are found in organs and at stages of development where chromoplasts are formed: petals, anthers and ripening fruits. Tobacco petals and fruits, which do not contain chromoplasts, show instead low levels of Pds/GUS expression. Transgenic tobacco seedlings were subjected to treatment with a range of inhibitors of carotenoid and chlorophyll biosynthesis. The results indicate that, in green tissues, carotenoid and chlorophyll levels are tightly co-regulated and that a chemically induced arrest in pigment biosynthesis results in activation of the Pds promoter. The promoter is also induced in etiolated seedlings, which contain much lower carotenoid levels than light-grown seedlings. These data suggest that in green tissues Pds gene transcription may respond to end-product regulation.