Bacterial phytopathogen Pectobacterium causes soft rot disease in several vegetable crops globally, resulting in heavy agricultural losses at both the pre and postharvest stages. The present work was carried out to screen Kimchi cabbage genetic resources conserved at the National Agrobiodiversity Center, Rural Development Administration, Korea, for resistance against the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum KACC 21701 over a period of three years (from 2020 to 2022). Infection of the phytopathogen was carried out at four-leaf stage and for each accession, twenty-five plants per germplasm were infected with KACC 21701. Kimchi cabbage cultivars Wangmatbaechu, Seoulbaechu, and CR Kiyoshi were used as control. Seven-days post-infection, the Disease Index (DI) values were manually recorded from zero to four, zero matched perfectly heathy plants and four completely dead plants. The 682 accessions of Kimchi cabbage exhibited varying degrees of disease resistance to KACC 21701 and thirty accessions, exhibiting a DI≤2, were considered for replication studies. During the replication studies, four landrace germplasms (IT102883, IT120036, IT120044, and IT120048) and one cultivar (IT187919) were confirmed to be moderately susceptible to KACC 21701. Results of the preliminary screening as well as replication studies were documented for the all the 682 germplasms. Addition of such information to the passport data of stored germplasms might serve as potential bio-resource for future breeders and researchers to develop resistant varieties or study the mechanisms involved in resistance of plants to such phytopathogen.

Riboswitches are structured RNA motifs that can directly bind specific metabolites. The binding of metabolites further regulates downstream metabolism eliminating the need for any regulatory proteins. We searched for novel bacterial vitamin B1 binding riboswitches in the metagenome of sun-dried saline soil. Soil microbial metagenomes were studied using NGS analysis. A total of approximately 50 Gb of the sequence data was obtained by Hi-seq and 454 GS FLX sequencing, and these sequences were subjected to riboswitch search. Hi-seq generated 614 contigs showing similarity to riboswitches, while 454-based sequencing generated 383 similar contigs. We matched whole metagenome contigs to local BLAST databases constructed using 91 previously known bacterial vitamin B1 thiamine pyrophosphate (TPP)-box motifs, and 11 SAM S-box motifs. Repetitive BLAST comparisons to local BLAST databases with nucleotide sequences from NGS identified 14 novel TPP-box motifs, and 7 S-box motifs respectively from the metagenome contigs. Further, RNA secondary structure analysis with public databases Rfam, and RibEx using these 21 riboswitch candidates revealed one contig, D8PYI to possess the most probable TPP-box structure. We constructed intragenic synthetic riboswitches to investigate whether the TPP-box motif region in D8PYI could harness gene expression in the presence of TPP. Construction of biosensors containing 100~400 bp fragments of D8PYI contigs, and in vivo imaging using the biosensors displayed TPP-specific changes in the expression of a green fluorescence protein reporter. In this regard, the adaptation of in silico riboswitch screening from environmental metagenomes could provide biosensors for detection of specific metabolites.

Plant-parasitic nematodes are the most devastating group of plant pathogens worldwide and are extremely challenging to control. In the present study, we have performed a genome wide analysis to identify common genes among four nematode species consisting of root-knot nematodes (Meloidogyne incognita and Meloidogyne hapla), cyst nematode (Heterodera glycines), and free living nematode (Caenorhabditis elegans) respectively. Using their whole genome sequences, we predicted 15,274 genes from M. incognita, 38,149 genes from M. hapla, 8,061 genes from H. glycines and 23,894 genes from C. elegans, where, among the predicted genes, 1,358, 1,350, 1,401, 1,365 respectively from each nematode, code for common groups of proteins. Further, 2,067, 2,086, 1,566, 2,903 genes were recollected using Clusters of Orthologous Groups (COG) database. Under our search criteria, a total of 800 common genes were identified in all the four studied nematode genomes. The most annotated conserved genes were obtained from four different species using Basic Local Alignment Searching Tool (BLAST). Uni- Prot Taxon identifier database was used to elucidate their taxonomic classification such as 698 genes under kingdom Metazoa, 660 genes confined to Nematoda, 290 genes in Chordata and 660 genes falling under class Chromadorea. The biochemical characterization of proteins expressed by these genes was examined using Pedant-Pro sequence analysis. The protein length, molecular weight, isoelectric point (pI), and transmembrane domain of the coded proteins were at a range of 300 to 999 amino acids (40.9%), molecular weight of over 100 kDa (96%), pI from 4.5 to 5.5 (27.6%) and 0 (56.6%), respectively. To classify protein function, the obtained BLAST hits were assigned to Gene Ontology classification scheme. The fractions of protein function were distributed as cellular component, biological processes and molecular function of the cell (22.2%), multicellular organism process (15.8%) and binding (48.3%), respectively. The current study provides an excellent resource for nematode functional genomics studies, which can be utilized further for studies on role of genes involved in nematode biological processes.

Human tissue-type plasminogen activator (t-PA) is responsible for fibrin-specific plasminogen activation and plays a key role in fibrinolysis thereby aiding breakdown of blood clots in the vasculature. In the present study, in order to develop a system for production of recombinant st-PA and t- PAHis6 proteins in transgenic rice seeds, a DNA fragment encoding t-PA gene was selected and cloned to a plant binary vector (pMJ21) harboring a rice GluB1 promoter, an N-terminal signal peptide of the rice glutelin B1 protein and a Pin II terminator. The constructed plasmid was transformed into Agrobacterium tumefaciens LBA4404 (pSB1) to facilitate introduction into rice callus. The insertion of the st-PA and t-PAHis6 genes into the genome of transgenic rice seeds and their transcripts were confirmed using PCR, and Southern blot as well as RT-PCR, respectively. The highest level of recombinant st-PA expression as determined by enzyme-linked immunosorbent assay (ELISA) was found to be 2,916 ng/total soluble protein (mg) in transgenic rice seeds. The amount of recombinant proteins expressed in transgenic plants was estimated to range from 634 ~ 2,916 ng/TSP mg (st-PA) and 925 ~ 2,640 ng/TSP mg(t- PAHis6), respectively. Immuno-blot analysis of transgenic rice seeds revealed single bands of approximately 68-kDa representing recombinant st-PA and t-PAHis6 proteins. These results demonstrate the expression and in vivo activity of recombinant st-PA and t-PAHis6 in transgenic rice seeds. This study is a promising endeavor for production of recombinant pharmaceutical proteins using rice seed system.