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Banana black leaf streak, caused by Mycosphaerella fijiensis M. Morelet, is a primary phytosanitary concern in export of this fruit around the world. To develop improved cultivars resistant to this disease, an understanding of host response to infection is necessary. In this study, we obtained expression data on 14,872 genes by microarray analysis in the resistant genotype Musa acuminata subsp. burmannicoides ‘Calcutta 4’ after inoculation with Mycosphaerella fijiensis. From these data, 16 genes were analyzed as potential reference genes and 12 genes were identified as potential early indicators of the onset of the host defense response. Subsequently, these genes were analyzed by quantitative reverse-transcription polymerase chain reaction in susceptible ‘Williams’ and resistant Calcutta 4. The 18S and 26S ribosomal subunit genes in both cultivars showed the best characteristics as reference genes. In all, 5 of the 12 defense genes expressed shortly after infection (peroxidase, pathogenesis-related [PR]-4, PR-10, phenylalanine ammonia-liase, and disease resistance response 1) showed overexpression in Calcutta 4 between 6 and 24 h after inoculation as opposed to Williams, which did not show overexpression after 144 h. Early induction of defense-related genes could be a key component of the resistance of the Calcutta 4 genotype against M. fijiensis. In addition, these five genes could be used as indicators of the activation of defense responses in the interaction between banana and M. fijiensis.

Electrospray mass spectrometry was used to accurately measure the molecular masses of single chain lectins from legume seeds and also of three recombinant lectins, expressed in Escherichia coli. The five single chain lectins, Erythrina corallodendron lectin, soybean and peanut agglutinins, Dolichos biflorus lectin, and Phaseolus vulgaris hemagglutinin E, all showed evidence of C-terminal proteolytic processing, in some cases to "ragged" ends, when their masses were compared to those expected from their cDNA sequences and their known carbohydrate chains. Recombinant forms of the lectins from E. corallodendron, soybean, and peanut also showed C-terminal trimming, but not to the same points as the natural forms. Discrepancies between the protein and cDNA sequences of the E. corallodendron lectin were resolved by combined liquid chromatography-mass spectrometry peptide mapping and protein sequencing experiments, and the presence of a second glycosylation site was demonstrated. Our data show that all of these lectins undergo C-terminal proteolytic processing of a readily attacked peptide segment. This trimming is frequently imprecise, and the resulting heterogeneity may be a major contributor to the appearance of isolectin forms of these proteins.

Electrospray mass spectrometry was used to accurately measure the molecular masses of single chain lectins from legume seeds and also of three recombinant lectins, expressed in Escherichia coli. The five single chain lectins, Erythrina corallodendron lectin, soybean and peanut agglutinins, Dolichos biflorus lectin, and Phaseolus vulgaris hemagglutinin E, all showed evidence of C-terminal proteolytic processing, in some cases to "ragged" ends, when their masses were compared to those expected from their cDNA sequences and their known carbohydrate chains. Recombinant forms of the lectins from E. corallodendron, soybean, and peanut also showed C-terminal trimming, but not to the same points as the natural forms. Discrepancies between the protein and cDNA sequences of the E. corallodendron lectin were resolved by combined liquid chromatography-mass spectrometry peptide mapping and protein sequencing experiments, and the presence of a second glycosylation site was demonstrated. Our data show that all of these lectins undergo C-terminal proteolytic processing of a readily attacked peptide segment. This trimming is frequently imprecise, and the resulting heterogeneity may be a major contributor to the appearance of isolectin forms of these proteins.

Examination of the three-dimensional structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin [(1991) Science 254, 862-866], revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134-Trp135, which can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound galactose. Comparison of the primary sequence of ECorL with that of soybean agglutinin, specific for galactose and its C-2 substituted derivatives, and of peanut agglutinin, specific for galactose only, showed that in soybean agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Ser-Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser-Glu- Tyr-Asn. Three mutants of ECorL were therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser-Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the double mutant L2; Y108T. They were expressed in Escherichia coli, as done for recombinant ECorL [(1992) Eur. J. Biochem. 205, 575-581]. The mutants had the same hemagglutinating activity as native or rECorL. Their specificity for galactose, GalNAc and Me beta GalNDns was examined by inhibition of hemagglutination and of the binding of the lectin to immobilized asialofetuin; in addition, their association constants with Me alpha GalNDns and Me beta GalNDns were measured by spectrofluorimetric titration. The results showed that Y108T had essentially similar specificity as the native and recombinant lectins. The affinity of L2 and L2;Y108T for galactose was also the same as ECorL, but they had a lower affinity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less). This appears to be largely due to steric hindrance by the two additional amino acids present in the cavity region in these mutants. Our findings also provide an explanation for the inability of PNA to accommodate C-2-substituted galactose derivatives at its primary subsite.

The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library. It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids. The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol. wt. 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein. The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose. The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes. Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose.

Most Agrobacterium-mediated transformation procedures described in the literature have proven to be inefficient for transformation of Andean potato cultivars. We describe here a more successful method for transformation of two of these cultivars, Diacol Capiro (DC) and Parda Pastusa (PP). Leaf explants were transformed using vector pBI121 carrying the β-glucuronidase and kanamycin resistance genes. Calli and shoot formation occurred after 5–8 weeks on a selection medium containing 3 mg/l zeatin and 1 mg/l indole acetic acid. The shoot regeneration frequency was 28% and 34% for DC and PP, respectively. Of the kanamycin-resistant plantlets obtained, 51% of those of DC and 13% of those of PP were confirmed to be transformants by histochemical assays, polymerase chain reaction analyses and Southern blotting techniques.