Enhanced protection against fungal attack by regulated expression of the b-32 gene in transgenic plants
--F. Forlani, L. Corazza, B. Balmas, G. Donini, G. Rotino, M. Motto and M. Maddaloni

Plants use a variety of defensive mechanisms against infections by insects, viruses, bacteria, and fungi. Several protective proteins and secondary metabolites are synthesized either constitutively or in an inducible fashion upon pathogen attack. Among these antimicrobial molecules, the ribosome inactivating proteins (RIPs) are produced sometimes in large amounts by species widely distributed throughout the plant kingdom. RIPs are severe inhibitors of eukaryotic ribosomes by enzymatic cleaving of a particular N-glycosyl bond in the 28S rRNA, so that the elongation factor 2 cannot properly bind to the ribosomes. RIPs are classified into two major classes. Type 1 ribosome-inactivating proteins, such as those of wheat and barley, consists of a single catalytic peptide (MW 30-32 kDa). Type 2 ribosome-inactivating proteins, such as ricin and abrin, are heterodimers composed of subunits A and B joined by a disulfide bond. The A chain is similar in size and enzymatic activity to type 1 RIPs. The B chain is a docking peptide promoting the transport of the holoprotein into the cell. The B chain increases the toxicity of type 2 RIPs to eukaryotic cells by a factor up to 106 in respect to type 1 RIPs.

In maize endosperm a cytosolic albumin with a molecular weight of 32 kDa, termed b-32, is synthesized in temporal and quantitative coordination with the deposition of storage proteins. The synthesis of b-32 is under the control of the regulatory genes Opaque2 (O2) and Opaque6 (O6), as the mutant alleles of these loci prevent its accumulation. While it has been clearly demonstrated that the protein product of the O2 locus is a strong transcriptional activator of the b-32 gene promoter, the relation between the b-32 and the O6 gene is not yet clarified.

In a previous paper we have noted that the DNA sequence of the b-32.66 cDNA is in part homologous to the protein-synthesis inhibitor II from dehusked barley grain (Lohmer et al., EMBO J. 10:617-624, 1991). Fifty-eight amino acids are conserved in a stretch of 173 residues. When the positions from amino acid 80 to amino acid 110 are considered, the identity reaches the value of 50%; of the remaining amino acid residues, 40% are similar.

We have produced transgenic tobacco plants with a construct (pwun-B32) expressing the b-32 gene under control of a promoter inducible by fungal attack. Some of the transgenics show enhanced resistance to Rhizoctonia solani AG4. The construct pwun-B32 was also used to transform Solanum tuberosum and Solanum integrifolium (wild aubergine). At present several kanamicin-resistant plants of both species have been obtained and we have preliminary results showing that the large majority of transgenic potato plants, under wounding conditions, produce a peptide immunoreactive to anti-b-32 antibodies.

Such a test has not been done for S. integrifolium. In the future we will challenge transgenic S. tuberosum and S. integrifolium plants with specific fungal strains to test if they have increased resistance as well. 

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