Expression of Acidothermus cellulolyticus E1 endo-beta-1,4-glucanase catalytic domain in transplastomic tobacco.

As part of an effort to develop transgenic plants as a system for the production of lignocellulose-degrading enzymes, we evaluated the production of the endo-beta-1,4-glucanase E1 catalytic domain (E1cd) of Acidothermus cellulolyticus in transplastomic tobacco. In an attempt to increase the translation efficiency of the E1cd cassette, various lengths of the N-terminus of the psbA gene product were fused to the E1cd protein. The psbA gene of the plastid genome encodes the D1 polypeptide of photosystem II and is known to encode an efficiently translated mRNA. Experiments in an Escherichia coli expression system indicated that the fusion of short (10-22 amino acid) segments of D1 to E1cd resulted in modest increases in E1cd abundance and were compatible with E1cd activity. Plastid expression cassettes encoding unmodified E1cd and a 10-amino-acid D1 fusion (10nE1cd) were used to generate transplastomic tobacco plants. Expression of the E1cd open reading frame in transplastomic tobacco resulted in very low levels of the enzyme. The transplastomic plants accumulated a high level of E1cd mRNA, however, indicating that post-transcriptional processes were probably limiting the production of recombinant protein. The accumulation of 10nE1cd in transplastomic tobacco was approximately 200-fold higher than that of unmodified E1cd, yielding 10nE1cd in excess of 12% of total soluble protein in the extracts of the lower leaves. Most importantly, the active recombinant enzyme was recovered very easily and efficiently from dried plant material and constituted as much as 0.3% of the dry weight of leaf tissue.

Sgt1, but not Rar1, is essential for the RB-mediated broad-spectrum resistance to potato late blight.

BACKGROUND: Late blight is the most serious potato disease world-wide. The most effective and environmentally sound way for controlling late blight is to incorporate natural resistance into potato cultivars. Several late blight resistance genes have been cloned recently. However, there is almost no information available about the resistance pathways mediated by any of those genes. RESULTS: We previously cloned a late blight resistance gene, RB, from a diploid wild potato species Solanum bulbocastanum. Transgenic potato lines containing a single RB gene showed a rate-limiting resistance against all known races of Phytophthora infestans, the late blight pathogen. To better understand the RB-mediated resistance we silenced the potato Rar1 and Sgt1 genes that have been implicated in mediating disease resistance responses against various plant pathogens and pests. The Rar1 and Sgt1 genes of a RB-containing potato clone were silenced using a RNA interference (RNAi)-based approach. All of the silenced potato plants displayed phenotypically normal growth. The late blight resistance of the Rar1 and Sgt1 silenced lines were evaluated by a traditional greenhouse inoculation method and quantified using a GFP-tagged P. infestans strain. The resistance of the Rar1-silenced plants was not affected. However, silencing of the Sgt1 gene abolished the RB-mediated resistance. CONCLUSION: Our study shows that silencing of the Sgt1 gene in potato does not result in lethality. However, the Sgt1 gene is essential for the RB-mediated late blight resistance. In contrast, the Rar1 gene is not required for RB-mediated resistance. These results provide additional evidence for the universal role of the Sgt1 gene in various R gene-mediated plant defense responses.

Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight.

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating potato disease in the world. Control of late blight in the United States and other developed countries relies extensively on fungicide application. We previously demonstrated that the wild diploid potato species Solanum bulbocastanum is highly resistant to all known races of P. infestans. Potato germplasm derived from S. bulbocastanum has shown durable and effective resistance in the field. Here we report the cloning of the major resistance gene RB in S. bulbocastanum by using a map-based approach in combination with a long-range (LR)-PCR strategy. A cluster of four resistance genes of the CC-NBS-LRR (coiled coil-nucleotide binding site-Leu-rich repeat) class was found within the genetically mapped RB region. Transgenic plants containing a LR-PCR product of one of these four genes displayed broad spectrum late blight resistance. The cloned RB gene provides a new resource for developing late blight-resistant potato varieties. Our results also demonstrate that LR-PCR is a valuable approach to isolate genes that cannot be maintained in the bacterial artificial chromosome system.