SUMMARY: In order to clone and analyse the avirulence gene AVR‐Pia from Japanese field isolates of Magnaporthe oryzae , a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95‐1, gained virulence towards the rice cultivar Aichi‐asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co‐segregated with avirulence towards Aichi‐asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR‐Pia . Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I–VI, and these were cloned individually into a hygromycin‐resistant vector for the transformation of the mutant Ina168m95‐1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR‐Pia . By deletion analysis of fragment V, AVR‐Pia was localized to an 1199‐bp DNA fragment, which included a 255‐bp open reading frame with weak homology to a bacterial cytochrome‐ c ‐like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co‐segregated with the AVR‐Pia locus in a genetic map that was constructed using Chinese isolates.

AVR-Pia, an avirulence gene in the genome of the rice blast fungus Magnaporthe oryzae, triggers a hypersensitive reaction in rice cultivars harbourin

The inulin fructotransferase (DFA III-forming)(EC 4.2.2.18) gene in Nonomuraea sp. ID06-A0189 was amplified from genomic DNA, sequenced and expressed in Escherichia coli. The 1326-bp gene, designated as Nsp-ift, encodes a protein composed of a putative 37-amino-acid signal peptide and 404-amino-acid mature protein. A putative ribosomal binding sequence was identified 12 bases upstream from the start codon. However, a typical bacterial promoter could not be found by in silico analysis. The deduced amino-acid sequence of the enzyme was most similar to that of inulin fructotransferase (DFA I-forming) in Frankia sp. EAN1pec. Phylogenetic analysis of deduced amino-acid sequences indicated that Nonomuraea sp. ID06-A0189 and Frankia sp. EAN1pec inulin fructotransferases formed a distinct clade from those from Arthrobacter sp. H65-7, A. globiformis and Bacillus sp. snu-7 that showed 57, 56 and 56% identity to that of Nsp-ift, respectively. The Nsp-ift without a putative signal peptide was successfully expressed in E. coli and partially purified using His-tag affinity chromatography. The recombinant enzyme displayed optimum temperature between 65 and 70 °C, optimum pH between 5.5 and 6.0 and remained stable up to 70 °C. The properties were identical to those of the original enzyme. Of 10 Nonomuraea species tested by Southern hybridization, enzyme activity measurements and PCR, only Nonomuraea sp. ID06-A0189 has the Nsp-ift gene, suggesting that Nsp-ift is not highly conserved in this genus.

Lactic acid bacteria (LAB) might switch the Th2 biased immune response in allergic patients towards a balanced Th1/Th2 immune profile, leading to amelioration of allergy. To select strains of LAB that could be of potential application for foods in controlling allergy, 35 bacterial strains were screened in vitro using murine splenocytes and peritoneal exudate cells (PECs). Streptococcus thermophilus AHU1838 (FERM AP-21009), and Lactobacillus paracasei subsp. casei AHU1839 (FERM AP-21010) enhanced the secretion of Th1 cytokines such as interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). The two strains of LAB also up-regulated the expression of CD40, and CD86 in dendritic cells (DCs), and activated cytotoxic T lymphocytes (CTL). These two strains could therefore be used in producing fermented food products that can enhance the Th1 immune profile which is important in ameliorating allergy.