| The objective of this study was to test the hypothesis that resistance to Rhizoctonia root rot, caused by Rhizoctonia solani AG8 and R. oryzae, could be obtained in barley by introducing a 42-kDa endochitinase gene from Trichoderma harzianum into the barley genome. The Trichoderma endochitinase gene cThEn42 (1272bp) was modified to increase its G+C content from 53.3% to 65.1%, to enhance expression in barley. Transformants of the yeast Pichia pastoris with the gene [cThEn42( GC)) synthesized and secreted into the culture medium active endochitinase of T. harzianum. Purified recombinant endochitinase was highly inhibitory to in-vitro growth of both R. solani AG8 and R. oryzae, and less inhibitory to that of Gaeumannomyces graminis var. tritici. Growth of Fusarim graminearum, F. pseudograminearum, and F. culmorum was not inhibited. Polyclonal antibodies reacted specifically with the purified endochitinase produced. Eight double-cassette vectors containing cThEn42(GC) were constructed for Agrobacterium -mediated transformation of barley. Of the primary transformants, 22 synthesized recombinant enzyme in leaves and roots with the gene under the control of a pUbi-1 promoter and 23 with the gene driven with a CaMV 35S promoter. T1 seedlings of eight different transformants carrying the transgene under the control of the ubiquitin promoter and four different signal peptides were screened twice for resistance to each of the Rhizoctonia species using a seedling assay with infested rooting medium. In both tests, seedlings of at least six of the eight progenies segregated were distinctly healthier phenotypes than the untransformed Golden Promise control, as indicated by reduced or no leaf chlorosis (typical of severe Rhizoctonia root rot) and greater seedling height. In addition, air-dry weights of the roots were significantly greater (P = 0.01) in more seedlings of the T1 progeny from one or more of the primary transformants compared to the untransformed controls. Based on these three phenotypes of less chlorosis, taller seedling, and greater root weight, all six of the eight T1 progenies were judged to have transgenic plants with resistance to both pathogens. Of these, progeny from a transformant with barley chitinase-26 and the fungal signal and activation peptide [HVChi-FSP-FAP] contained 9/12 seedlings with a highly resistant phenotype. |
