Increased resistance to fusarium wilt in transgenic tobacco lines co-expressing chitinase and wasabi defensin genes

Marker-free transgenic tobacco (Nicotiana tabacum) lines containing a chitinase (ChiC) gene isolated from Streptomyces griseus strain HUT 6037 were produced by Agrobacterium-mediated transformation. One marker-free transgenic line, TC-1, was retransformed with the wasabi defensin (WD) gene, isolated from Wasabia japonica. Of the retransformed shoots, 37% co-expressed the ChiC/WD genes, as confirmed by western and northern analyses. Southern blot analysis showed that no chromosomal rearrangement was introduced between the first and the second transformation. Transgenic lines either expressing ChiC or WD, or co-expressing both genes were challenged with Fusarium oxysporum f.sp. nicotianae (Fon). Assessment of in vitro plant survival in the presence of Fon showed that transgenic lines co-expressing both genes had significantly enhanced protection against the fungus (infection indices 0 center dot 0-1.center dot 2) compared with corresponding isogenic lines expressing either of the genes (infection indices 2 center dot 5-9 center dot 8). Whole-plant infection indices in transgenic lines were significantly related (r = 0 center dot 93, P < 0 center dot 01) to the extent of root colonization of the host, which ranged from 2 center dot 1% to 11 center dot 3% in lines co-expressing both genes, and from 16 center dot 8% to 37 center dot 7% in lines expressing just one of the genes (compared with 86 center dot 4% in non-transformed controls). Leaf extracts of transgenic lines also inhibited mycelial growth of Fon in vitro and caused hyphal abnormalities.