Mature-stem expression of a silencing-resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane.

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally-controlled expression of a silencing-resistant gene encoding a vacuole-targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole-cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high-isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value-added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing-resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field-grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale.

Different domains of Phytophthora sojae effector Avr4/6 are recognized by soybean resistance genes Rps4 and Rps6.

At least 12 avirulence genes have been genetically identified and mapped in Phytophthora sojae, an oomycete pathogen causing root and stem rot of soybean. Previously, the Avr4 and Avr6 genes of P. sojae were genetically mapped within a 24 kb interval of the genome. Here, we identify Avr4 and Avr6 and show that they are actually a single gene, Avr4/6, located near the 24-kb region. Avr4/6 encodes a secreted protein of 123 amino acids with an RXLR-dEER protein translocation motif. Transient expression of Avr4/6 in soybean leaves revealed that its gene product could trigger a hypersensitive response (HR) in the presence of either Rps4 or Rps6. Silencing Avr4/6 in P. sojae stable transformants abolished the avirulence phenotype exhibited on both Rps4 and Rps6 soybean cultivars. The N terminus of Avr4/6, including the dEER motif, is sufficient to trigger Rps4-dependent HR while its C terminus is sufficient to trigger Rps6-mediated HR. Compared with alleles from avirulent races, alleles of Avr4/6 from virulent races possess nucleotide substitutions in the 5' untranslated region of the gene but not in the protein-coding region.

Identification and occurrence of the LTR-Copia-like retrotransposon, PSCR and other Copia-like elements in the genome of Phytophthora sojae.

Sequence analysis of the genomic region of Phytophthora sojae close to the Avr4/6 locus specifying virulence on soybean identified a Ty1/Copia-like retrotransposon that we have named Phytophthora sojae Copia-like retrotransposon (PSCR). Twelve near-complete homologs of PSCR were found in the published P. sojae genome sequence, none of which encoded a full-length polyprotein characteristic of Copia-like retrotransposons, or appears to exhibit transcriptional activity or show evidence of recent movement, suggesting they are non-functional and unlikely to have caused pathogenic variability. However, reconstructed consensus PSCR sequence encoding a full-length polyprotein resembles a functional, ancestral retroelement within P. sojae. Homologs were also found in sequence databases of other Phytophthora species. Database searches found other families of Copia-like elements in genomes of P. sojae, P. ramorum and P. infestans that were different from members of the PSCR family and from Copia-like elements reported in other organisms. It is possible that the various families of Copia-like retroelements identified in this study represent introgressions into the genome of ancient ancestor(s) of current Phytophthora species, where they have evolved and diverged considerably during the speciation. Some Copia-like families are transcriptionally active with the potential to transpose and contribute to pathogenic variation in current populations of P. sojae.

Phytophthora sojae avirulence genes Avr4 and Avr6 are located in a 24kb, recombination-rich region of genomic DNA.

A cross between two different races (race 7xrace 25) of the soybean root and stem rot pathogen Phytophthora sojae was analyzed to characterize the genomic region flanking two cosegregating avirulence genes, Avr4 and Avr6. Both genes cosegregated in the ratio of 82:17 (avirulent:virulent) in an F(2) population, suggestive of a single locus controlling both phenotypes. A chromosome walk was commenced from RAPD marker OPE7.1C, 2.0cM distant from the Avr4/6 locus. Three overlapping cosmids were isolated which included genetic markers that flank the Avr4/6 locus. The chromosome walk spanned a physical distance of 67kb which represented a genetic map distance of 22.3cM, an average recombination frequency of 3.0kb/cM and 11.7-fold greater than the predicted average recombination frequency of 35.3kb/cM for the entire P. sojae genome. Six genes (cDNA clones) expressed from the Avr4/6 genomic region encompassed by the cosmid contig were identified. Single nucleotide polymorphisms and restriction fragment length polymorphisms showed these six genes were closely linked to the Avr4/6 locus. Physical mapping of the cDNA clones within the cosmid contig made it possible to deduce the precise linkage order of the cDNAs. None of the six cDNA clones appear to be candidates for Avr4/6. We conclude that two of these cDNA clones flank a physical region of approximately 24kb and 4.3cM that appears to include the Avr4/6 locus.