Satyabrata Nanda

Molecular characterization and heterologous expression of a pathogen induced PR5 gene from garlic ( Allium sativum L.) conferring enhanced resistance to necrotrophic fungi

Pathogenesis-related protein-5 (PR5) is encoded by a complex group of gene family that are involved in host defense against biotic and abiotic stresses as well as regulation of physiological processes in a wide range of plants and animals. In the present study, we isolated and characterized a PR5 gene, designated as AsPR5, induced in response to Fusarium oxysporum f. sp. cepae (FOC) infection in garlic (Allium sativum). AsPR5 cDNA encoded a protein of 223 amino acids including a 22 amino acid signal peptide at the N-terminus suggesting that it is an apoplast secreted acidic protein. AsPR5 protein contains 16 conserved cysteine residues and five additional conserved amino acids- an arginine, a glutamic acid and three aspartic acids related with antifungal activity of most plant thaumatin like proteins. Semi-quantitative RT-PCR showed that the transcript levels of AsPR5 was higher in stem tissues, the primary site of FOC infection, followed by leaves, roots and flowers. Temporal expression analysis using qPCR revealed high expression of AsPR5 upon infection with FOC as well as various phytohormones (JA, ET and ABA) and stress stimuli (wounding, high temperature and salinity) indicating its involvement in both biotic and abiotic stresses. Analyses of purified recombinant protein against different fungal pathogens showed improved antifungal activity compared to other reported PR5 proteins. Furthermore, the Arabidopsis plants ectopically expressing AsPR5 showed enhanced resistance to fungal pathogen, Botrytis cinerea and constitutively higher expression of defense responsive genes such as Lox3, PDF1.2, PAD3, AOS, and AOC. These results suggest that, besides antifungal activities against the necrotrophs, AsPR5 also play significant role in activating multiple defense pathways for enhancing stress resistance in crop plants.

Multiple garlic (Allium sativum L.) microRNAs regulate the immunity against the basal rot fungus Fusarium oxysporum f. sp. Cepae

The basal plate rot fungus, Fusarium oxysporum f. sp. cepae (FOC), is the most devastating pathogen posing a serious threat to garlic (Allium sativum L.) production worldwide. MicroRNAs (miRNAs) are key modulators of gene expression related to development and defense responses in eukaryotes. However, the miRNA species associated with garlic immunity against FOC are yet to be explored. In the present study, a small RNA library developed from FOC infected resistant garlic line was sequenced to identify immune responsive miRNAs. Forty-five miRNAs representing 39 conserved and six novel sequences responsive to FOC were detected. qRT-PCR analyses further classified them into three classes based on their expression patterns in susceptible line CBT-As11 and in the resistant line CBT-As153. North-blot analyses of six selective miRNAs confirmed the qRT-PCR results. Expression studies on a selective set of target genes revealed a negative correlation with the complementary miRNAs. Furthermore, transgenic garlic plant overexpresing miR164a, miR168a and miR393 showed enhanced resistance to FOC, as revealed by decreased fungal growth and up-regulated expression of defense-responsive genes. These results indicate that multiple miRNAs are involved in garlic immunity against FOC and that the overexpression of miR164a, miR168a and miR393 can augment garlic resistance to Fusarium basal rot infection.

Regulation of miR394 in Response to Fusarium oxysporum f. sp. cepae (FOC) Infection in Garlic (Allium sativum L)

MicroRNAs (miRNAs) are a class of post-transcriptional regulators that negatively regulate gene expression through target mRNA cleavage or translational inhibition and play important roles in plant development and stress response. In the present study, six conserved miRNAs from garlic (Allium sativum L.) were analyzed to identify differentially expressed miRNAs in response to Fusarium oxysporum f. sp. cepae (FOC) infection. Stem-loop RT-PCR revealed that miR394 is significantly induced in garlic seedlings post-treatment with FOC for 72 h. The induction of miR394 expression during FOC infection was restricted to the basal stem plate tissue, the primary site of infection. Garlic miR394 was also upregulated by exogenous application of jasmonic acid. Two putative targets of miR394 encoding F-box domain and cytochrome P450 (CYP450) family proteins were predicted and verified using 5′ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends) assay. Quantitative RT-PCR showed that the transcript levels of the predicted targets were significantly reduced in garlic plants exposed to FOC. When garlic cultivars with variable sensitivity to FOC were exposed to the pathogen, an upregulation of miR394 and down regulation of the targets were observed in both varieties. However, the expression pattern was delayed in the resistant genotypes. These results suggest that miR394 functions in negative modulation of FOC resistance and the difference in timing and levels of expression in variable genotypes could be examined as markers for selection of FOC resistant garlic cultivars.

Mining, characterization and validation of EST derived microsatellites from the transcriptome database of Allium sativum L

Expressed Sequence Tags (ESTs) with comprehensive transcript information are valuable resources for development of molecular markers as they are derived from conserved genic regions. The present study highlights the mining of EST database to deduce the class I hyper variable SSRs in A. sativum. From 21694 garlic EST sequences, 642 non-redundant SSRs were identified with an average frequency of 1 per 14.9 kb of garlic transcriptome. The most abundant SSR motifs were the mononucleotides (32.86%) followed by trinucleotides (28.50%) and dinucleotides (13.39%). Among the individual SSRs, (A/T)n accounted for the highest number (137; 21.33%) followed by (G/C)n (74; 11.52%) and (AAG)n (63;9.81%). Primers designed from a robust set of 7 AsESTSSRs resulted in the amplification of 63 polymorphic alleles in 14 accessions of garlic. The resolving power of the markers varied from 4.286 (AsSSR7) to 18.143 (AsSSR13) while the average marker index (MI) was 5.087. These EST-SSRs markers for garlic could be useful for the improvement of garlic linkage map and could be used for evaluating genetic variation and comparative genomics studies in Allium species.

Molecular modeling and docking characterization of CzR1, a CC-NBS-LRR R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum.

Plant NBS-LRR R-genes recognizes several pathogen associated molecular patterns (PAMPs) and limit pathogen infection through a multifaceted defense response. CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene isolated from Curcuma zedoaria L exhibit constitutive resistance to different strains of P. aphanidermatum. Majority of the necrotrophic oomycetes are characterized by the presence of carbohydrate PAMPs β-glucans in their cell walls which intercat with R-genes. In the present study, we predicted the 3D (three dimensional) structure of CzR1 based on homology modeling using the homology module of Prime through the Maestro interface of Schrodinger package ver 2.5. The docking investigation of CzR1 with β-glucan using the Glide software suggests that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 act as catalytic residues and are involved in hydrogen bonding with ligand β-(1,3)-D-Glucan. The calculated distance between the carboxylic oxygen atoms of Glu187–Asp355 pair is well within the distance of 5Å suggesting a positive glucanase activity of CzR1. Elucidation of these molecular characteristics will help in in silico screening and understanding the structural basis of ligand binding to CzR1 protein and pave new ways towards a broad spectrum rhizome rot resistance development in the cultivated turmeric.

Development of a sequence-tagged site (STS) marker for sex identification in the dioecious rattan species Calamus guruba Buch.-Ham.

Calamus guruba Buch.-Ham., a perennial, dioecious rattan species of the family Arecaceae has recently emerged as an important source of tribal economy owing to its high-quality flexible canes for furniture manufacturing and cottage industries. The dioecious nature together with disproportionate distribution of male and female sex organs has been the major constraint in the development of an effective breeding programme for the plant. Early identification of the male and female genotypes at the seedling stage is a pre-requisite to ensure genetic improvement of C. guruba. In the present study, we used 30 inter simple sequence repeat (ISSR) markers to develop a sequence-tagged site (STS) linked to male sex in C. guruba. Molecular analysis of bulked DNA pooled from male and female genotypes resulted in the isolation of a putative male-specific marker CgMSM. Partial sequencing of CgMSM resulted in an STS marker CgMY1 which could successfully amplify a 597-bp fragment in male but not in the female plants. DNA gel blot analysis confirmed it as a single-copy locus in the male genome of C. guruba. Further validation of CgMY1 in the natural population of C. guruba resulted in precise detection of 9 males and 6 females from 15 individuals with unknown sex. Therefore, the STS marker CgMY1 could be used as a proficient tool for early sex differentiation and form the basis of a sustainable breeding programme for genetic improvement of C. guruba.

Identification of Novel Source of Resistance and Differential Response of Allium Genotypes to Purple Blotch Pathogen, Alternaria porri (Ellis) Ciferri

Purple blotch, caused by Alternaria porri (Ellis) Cifferi, is a serious disease incurring heavy yield losses in the bulb and seed crop of onion and garlic worldwide. There is an immediate need for identification of effective resistance sources for use in host resistance breeding. A total of 43 Allium genotypes were screened for purple blotch resistance under field conditions. Allium cepa accession ‘CBT-Ac77’ and cultivar ‘Arka Kalyan’ were observed to be highly resistant. In vitro inoculation of a selected set of genotypes with A. porri, revealed that 7 days after inoculation was suitable to observe the disease severity. In vitro screening of 43 genotypes for resistance to A. porri revealed two resistant lines. An additional 14 genotypes showed consistent moderate resistance in the field as well as in vitro evaluations. Among the related Allium species, A. schoenoprasum and A. roylei showed the least disease index and can be used for interspecific hybridization with cultivated onion. Differential reaction analysis of three A. porri isolates (Apo-Chiplima, Apn-Nasik, Apg-Guntur) in 43 genotypes revealed significant variation among the evaluated Allium species (P = 0.001). All together, the present study suggest that, the newly identified resistance sources can be used as potential donors for ongoing purple blotch resistance breeding program in India.

Molecular cloning and expression analysis of four turmeric MAP kinase genes in response to abiotic stresses and phytohormones

Plant mitogen activated protein kinase (MAPK) cascades comprise a complex network playing a major role in regulating extracellular stimuli as well as developmental processes. The present study involves cloning four MAPKs (ClMPK1, 3, 4 and 5) from Curcuma longa. All four ClMPKs have fully canonical motifs of MAPK and each is represented by a single copy in the turmeric genome. The analysis of exon-intron junctions revealed conserved nature of ClMPKs across different plant groups. The RT-qPCR analysis showed their expression in mature plant tissues. The transcript analysis using the RT-qPCR shows that the four ClMPKs were differentially regulated by cold, salinity, and drought stresses. ClMPK4 showed a significant upregulation in the presence of NaCl, polyethylene glycol, and mannitol. The time-course expression analysis revealed a marked accumulation of ClMPK1 and ClMPK4 transcripts after mechanical wounding or applications of abscisic acid, H2O2, methyl jasmonate, and salicylic acid. ClMPK5 showed a unique and pronounced expression in response to hexavalent chromium (CrVI).

In silico analysis of onion chitinases using transcriptome data

Chitinases are glycoside hydrolase (GH) family of proteins having multifaceted roles in plants. It is of interest to identify and characterize chitinase-encoding genes from the popular bulbous plant onion (Allium cepa L.). We have used the EST sequences for onion chitinases to elucidate its functional features using sequence, structure and functional analysis. These contigs belong to the GH19 chitinases family according to domain architecture analysis. They have highly conserved chitinase motifs including motifs exclusive to plant chitinases as implied using the MEME based structural characterization. Estimation of biochemical properties suggested that these proteins have features to form stable and hydrophilic proteins capable of localizing extracellular and in vacuoles. Further, they have multiple cellular processes including defense role as inferred by DeepGO function prediction. Phylogenetic analysis grouped them as class I and class VII plant chitinase, with possible abundance of class I chitinase in onion. These observations help in the isolation and functional validation of onion chitinases.