Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes-aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)-in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.

Plant chitinases have been known as pathogenesis-related (PR) proteins, but recent studies suggest that they play functional roles during normal plant growth and development. We previously isolated two cDNA clones encoding endochitinases, EuNOD-CHT1 and .CHT2, from the root nodules of Elaeagnus umbellata. These genes show differential expression patterns, with the EuNOD-CHT1 gene being active in the root nodules and meristems, while EuNOD-CHT2 is preferentially expressed in the infected cells of those nodules. To elucidate the functional roles of these two endochitinases, we have now constitutively expressed each gene in a heterologous plant system, Arabidopsis thaliana. Stable inheritance and expression of the transgenes were confirmed by genomic Southern hybridization and RT-PCR. Our transgenic plants did not differ morphologically from the wild types. However, constitutive expression of EuNOD-CHT1 and -CHT2 in Arabidopsis resulted in increased resistance against a fungal pathogen, Botrytis cinerea, but not against a bacterial agent, Pseudomonas syringae pv. Tomato DC3000. Expression levels were enhanced by both wounding and jasmonic acid treatments (for EuNOD-CHT1), or by jasmonic acid only (for EuNOD-CHT2). These data suggest that EuNOD-CHT1 and -CfiT2 primarily play defensive roles during root nodule development in E. umbellata.