Raj Kumar Joshi

Perspectives of genomic diversification and molecular recombination towards R-gene evolution in plants

Plants are under strong evolutionary pressure in developing new and noble R genes to recognize pathogen avirulence (avr) determinants and bring about stable defense for generation after generations. Duplication, sequence variation by mutation, disparity in the length and structure of leucine rich repeats etc., causes tremendous variations within and among R genes in a plant thereby developing diverse recognitional specificity suitable enough for defense against new pathogens. Recent studies on genome sequencing, diversity and population genetics in different plants have thrown new insights on the molecular evolution of these genes. Tandem and segmental duplication are important factors in R gene abundance as inferred from the distribution of major nucleotide binding site-leucine rich repeats (NBS-LRRs) type R-genes in plant genomes. Likewise, R-gene evolution is also thought to be facilitated by cluster formation thereby causing recombination and sequence exchange and resulting in haplotypic diversity. Population studies have further proven that balancing selection is responsible for the maintenance of allelic diversity in R genes. In this review, we emphasize and discuss on improved perspectives towards the molecular mechanisms and selection pressure responsible for the evolution of NBS-LRR class resistance genes in plants.

Mining and characterization of EST derived microsatellites in Curcuma longa L.

Turmeric (Curcuma longa L.) (Family: Zingiberaceae) is a perennial rhizomatous herbaceous plant often used as a spice since time immemorial. Turmeric plants are also widely known for its medicinal applications. Recently EST‐derived SSRs (Simple sequence repeats) are a free by‐product of the currently expanding EST (Expressed Sequence Tag) databases. SSRs have been widely applied as molecular markers in genetic studies. Development of high throughput method for detection of SSRs has given a new dimension in their use as molecular markers. A software tool SciRoKo was used to mine class I SSR in Curcuma EST database comprising 12953 sequences. A total of 568 non‐redundant SSR loci were detected with an average of one SSR per 14.73 Kb of EST. Furthermore, trinucleotide was found to be the most abundant repeat type among 1‐6‐nucleotide repeat types. It accounted for 41.19% of the total, followed by the mononucleotide (20.07%) and hexanucleotide repeats (15.14%). Among all the repeat motifs, (A/T)n accounted for the highest proportion followed by (AGG)n. These detected SSRs can be greatly used for designing primers that can be used as markers for constructing saturated genetic maps and conducting comparative genomic studies in different Curcuma species.

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.

Transcriptome profiling of the floral buds and discovery of genes related to sex-differentiation in the dioecious cucurbit Coccinia grandis (L.) Voigt

Dioecious species offer an inclusive structure to study the molecular basis of sexual dimorphism in angiosperms. Despite having a small genome and heteromorphic sex chromosomes, Coccinia grandis is a highly neglected dioecious species with little information available on its physical state, genetic orientation and key sex-defining elements. In the present study, we performed RNA-Seq and DGE analysis of male (MB) and female (FB) buds in C. grandis to gain insights into the molecular basis of sex determination in this plant. De novo assembly of 75 million clean reads resulted in 72,479 unigenes for male library and 63,308 unigenes for female library with a mean length of 736bp. 61,458 (85.57%) unigenes displayed significant similarity with protein sequences from publicly available databases. Comparative transcriptome analyses revealed 1410 unigenes as differentially expressed (DEGs) between MB and FB samples. A consistent correlation between the expression levels of DEGs was observed for the RNA-Seq pattern and qRT-PCR validation. Functional annotation showed high enrichment of DEGs involved in phytohormone biosynthesis, hormone signaling and transduction, transcriptional regulation and methyltransferase activity. High induction of hormone responsive genes such as ARF6, ACC synthase1, SNRK2 and BRI1-associated receptor kinase 1 (BAK1) suggest that multiple phytohormones and their signaling crosstalk play crucial role in sex determination in this species. Beside, the transcription factors such as zinc fingers, homeodomain leucine zippers and MYBs were identified as major determinants of male specific expression. Moreover, the detection of multiple DEGs as the miRNA target site implies that a small RNA mediated gene silencing cascade may also be regulating gender differentiation in C. grandis. Overall, the present transcriptome resources provide us a large number of DEGs involved in sex expression and could form the groundwork for unravelling the molecular mechanism of sex determination in C. grandis.

Molecular Cloning, Characterization, and Expression Analysis of Resistance Gene Candidates in Kaempferia galanga L.

Majority of the plant disease resistance genes expresses cytoplasmic receptor-like proteins characterized by an N-terminal nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain. Degenerative primers based on these conserved motifs were used to isolate NBS type sequences in Kaempferia galanga. Cloning and sequencing identified 12 Kaempferia NBS-type sequences called resistance gene candidates (RGCs) classified into four classes. The amino acid sequences of the RGCs detected the presence of conserved domains, viz., kinase-1a, kinase-2, and hydrophobic GLPL, categorizing them with the NBS–LRR class gene family. Structural and phylogenetic characterization grouped the RGCs with the non-toll interleukin receptor (non-TIR) subclasses of the NBS sequences. Reverse transcription PCR with 10 Kaempferia RGC specific primers revealed 7 out of 10 Kaempferia RGCs to be expressive. The isolation and characterization of Kaempferia RGCs has been reported for the first time in this study. This will provide a starting point towards characterization of candidate resistance genes in Kaempferia and can act as a source pool for disease resistance development in other asexually reproducing plants.

Characterization of mitogen activated protein kinases (MAPKs) in the Curcuma longa expressed sequence tag database

Mitogen activated protein kinase (MAPK) cascades are universal signal transduction modules that play crucial role in plant growth and development as well as biotic and abiotic stress responses. 20 and 17 MAPKs have been characterized in Arabidopsis and rice respectively, which are used for identification of the putative MAPKs in other higher plants. However, no MAPK gene sequences have yet been characterized for asexually reproducing plants. We describe the analysis of MAPK EST sequences from Curcuma longa (an asexually reproducible plant of great medicinal and economic significance). The four Curcuma MAPKs contains all 11 MAPK conserved domains and phosphorylation-activation motif, TEY. Phylogenetic analysis grouped them in the subgroup A and C as identified earlier for Arabidopsis. The Curcuma MAPKs identified showed high sequence homology to rice OsMPK3, OsMPK4 and OsMPK5 suggesting the presence of similar key element in signaling biotic and abiotic stress responses. Although further in vivo and in vitro analysis are required to establish the physiological role of Curcuma MAPKs, this study provides the base for future research on diverse signaling pathways mediated by MAPKs in Curcuma longa as well as other asexually reproducing plants.

Survey and characterization of NBS-LRR (R) genes in Curcuma longa transcriptome.

Resistance genes are among the most important gene classes for plant breeding purposes being responsible for activation of plant defense mechanisms. Among them, the nucleotide binding site-leucine rich repeat (NBS-LRR) class R-genes are the most abundant and actively found in all types of plants. Insilico characterization of EST database resulted in the detection of 28 NBS types R-gene sequences in Curcuma longa. All the 28 sequences represented the NB-ARC domain, 21 of which were found to have highly conserved motif characteristics and categorized as regular NBS genes. The Open Reading Frames varied from 361 (CL.CON.3566) to 112 (CL.CON.1267) with an average of 279 amino acids. Most alignment occurred with monocots (67.8%) with emphasis on Oryza sativa and Zingiber sequences. All best alignments with dicots occurred with Arabidopsis thaliana, Populus trichocarpa and Medicago sativa. These detected NBS type Rgenes from Curcuma longa can be used as a valuable resource for molecular marker development, molecular mapping of R-genes, and identification of resistance gene analogs and functional and evolutionary characterization of NBS–LRR–encoding resistance genes in asexually reproducing plants.

Exploiting EST databases for the mining and characterization of short sequence repeat (SSR) markers in Catharanthus roseus L.

Periwinkle (Catharanthus roseus L.) (Family: Apocyanaceae) is a ornamental plants with great medicinal properties. Although it is represented by seven species, little work has been carried out on its genetic characterization due to non-availability of reliable molecular markers. Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. With the rapid increase in the deposition of nucleotide sequences in the public databases and advent of bioinformatics tools, it has become a cost effective and fast approach to scan for microsatellite repeats and exploit the possibility of converting it into potential genetic markers. Expressed sequence tags (ESTs) from Catharanthus roseus were used for the screening of Class I (hyper variable) simple sequence repeats (SSRs). A total of 502 microsatellite repeats were detected from 21730 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs account to 1 SSR per 10.21 kb of EST. Mononucleotides was the most abundant class of microsatellite motifs. It accounted for 44.02% of the total, followed by the trinucleotide (26.09%) and dinucleotide repeats (14.34%). Among all the repeat motifs, (A/T)n accounted for the highest Proportion (36.25%) followed by (AAG)n. These detected SSRs can be used to design primers that have functional importance and should also facilitate the analysis of genetic diversity, variability, linkage mapping and evolutionary relationships in plants especially medicinal 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.