Heterologous expression and characterization of ToxA1 haplotype from India and its interaction with Tsn1 for spot blotch susceptibility in spring wheat.

BACKGROUND: ToxA, a necrotrophic effector protein, is present in the genome of fungal species like Parastagnospora nodorum, Pyrenophora tritici-repentis and Bipolaris sorokiniana. Tsn1 is the sensitivity gene in the host whose presence indicates more susceptibility to ToxA carrying pathogen, and ToxA-Tsn1 interaction follows an inverse gene-for-gene relationship. METHODS AND RESULTS: The present study involved cloning and expressing the ToxA1 haplotype from B. sorokiniana. It was found that the amplicon exhibited an expected product size of 471 bp. Sequence analysis of the ToxA1 nucleotide sequence revealed the highest identity, 99.79%, with P. tritici-repentis. The protein expression analysis showed peak expression at 16.5 kDa. Phylogenetic analysis of the ToxA1 sequence from all the Bipolaris isolates formed an independent clade along with P. tritici-repentis and diverged from P. nodorum. ToxA-Tsn1 interaction was studied in 18 wheat genotypes (11 Tsn1 and 7 tsn1) at both seedling and adult stages, validating the inverse gene-for-gene relationship, as the toxin activity was highest in the K68 genotype (Tsn1) and lowest in WAMI280 (tsn1). CONCLUSION: The study indicates that the haplotype ToxA1 is prevailing in the Indian population of B. sorokiniana. It would be desirable for wheat breeders to select genotypes with tsn1 locus for making wheat resistant to spot blotch.

Human beta casein fragment (54-59) modulates M. bovis BCG survival and basic transcription factor 3 (BTF3) expression in THP-1 cell line.

Immunostimulatory peptides potentiate the immune system of the host and are being used as a viable adjunct to established therapeutic modalities in treatment of cancer and microbial infections. Several peptides derived from milk protein have been reported to induce immunostimulatory activity. Human ß -casein fragment (54-59), natural sequence peptide (NS) carrying the Val-Glu-Pro-Ile-Pro-Tyr amino acid residues, was reported to activate the macrophages and impart potent immunostimulatory activity. In present study, we found that this peptide increases the clearance of M. bovis BCG from THP-1 cell line in vitro. The key biomolecules, involved in the clearance of BCG from macrophage like, nitric oxide, pro-inflammatory cytokines and chemokines, were not found to be significantly altered after peptide treatment in comparison to the untreated control. Using proteomic approach we found that BTF3a, an isoform of the Basic Transcription Factor, BTF3, was down regulated in THP-1 cell line after peptide treatment. This was reconfirmed by real time RT-PCR and western blotting. We report the BTF3a as a novel target of this hexapeptide. Based on the earlier findings and the results from the present studies, we suggest that the down regulation of BTF3a following the peptide treatment may augment the M. bovis BCG mediated apoptosis resulting in enhanced clearance of M. bovis BCG from THP-1 cell line.