Jolly Basak

Plant–Pathogen Interactions: What Microarray Tells About It?

Plant defense responses are mediated by elementary regulatory proteins that affect expression of thousands of genes. Over the last decade, microarray technology has played a key role in deciphering the underlying networks of gene regulation in plants that lead to a wide variety of defence responses. Microarray is an important tool to quantify and profile the expression of thousands of genes simultaneously, with two main aims: (1) gene discovery and (2) global expression profiling. Several microarray technologies are currently in use; most include a glass slide platform with spotted cDNA or oligonucleotides. Till date, microarray technology has been used in the identification of regulatory genes, end-point defence genes, to understand the signal transduction processes underlying disease resistance and its intimate links to other physiological pathways. Microarray technology can be used for in-depth, simultaneous profiling of host/pathogen genes as the disease progresses from infection to resistance/susceptibility at different developmental stages of the host, which can be done in different environments, for clearer understanding of the processes involved. A thorough knowledge of plant disease resistance using successful combination of microarray and other high throughput techniques, as well as biochemical, genetic, and cell biological experiments is needed for practical application to secure and stabilize yield of many crop plants. This review starts with a brief introduction to microarray technology, followed by the basics of plant–pathogen interaction, the use of DNA microarrays over the last decade to unravel the mysteries of plant–pathogen interaction, and ends with the future prospects of this technology.

Molecular marker-assisted genotyping of mungbean yellow mosaic India virus resistant germplasms of mungbean and urdbean.

Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus begomovirus causes the yellow mosaic disease in a number of economically important edible grain legumes including mungbean (Vigna radiata), urdbean (Vigna mungo) and soybean (Glycine max). The disease is severe, critical, open spread and inflicts heavy yield losses annually. The objective of this study is to develop molecular markers linked to MYMIV-resistance to facilitate genotyping of urdbean and mungbean germplasms for MYMIV-reaction. Resistance-linked molecular markers were successfully developed from consensus motifs of other resistance (R) gene or R gene homologue sequences. Applying linked marker-assisted genotyping, plant breeders can carry out repeated genotyping throughout the growing season in absence of any disease incidence. Two MYMIV-resistance marker loci, YR4 and CYR1, were identified and of these two CYR1 is completely linked with MYMIV-resistant germplasms and co-segregating with MYMIV-resistant F2, F3 progenies of urdbean. The present study demonstrated that these two markers could be efficiently employed together in a multiplex-PCR-reaction for genotyping both V. mungo and V. radiata germplasms from field grown plants and also directly from the seed stock. This method of genotyping would save time and labour during the introgression of MYMIV-resistance through molecular breeding, as methods of phenotyping against begomoviruses are tedious, labour and time intensive.

The Phylogeny and Biogeographic History of Ashes (Fraxinus, Oleaceae) Highlight the Roles of Migration and Vicariance in the Diversification of Temperate Trees

The cosmopolitan genus Fraxinus, which comprises about 40 species of temperate trees and shrubs occupying various habitats in the Northern Hemisphere, represents a useful model to study speciation in long-lived angiosperms. We used nuclear external transcribed spacers (nETS), phantastica gene sequences, and two chloroplast loci (trnH-psbA and rpl32-trnL) in combination with previously published and newly obtained nITS sequences to produce a time-calibrated multi-locus phylogeny of the genus. We then inferred the biogeographic history and evolution of floral morphology. An early dispersal event could be inferred from North America to Asia during the Oligocene, leading to the diversification of the section Melioides sensus lato. Another intercontinental dispersal originating from the Eurasian section of Fraxinus could be dated from the Miocene and resulted in the speciation of F. nigra in North America. In addition, vicariance was inferred to account for the distribution of the other Old World species (sections Sciadanthus, Fraxinus and Ornus). Geographic speciation likely involving dispersal and vicariance could also be inferred from the phylogenetic grouping of geographically close taxa. Molecular dating suggested that the initial divergence of the taxonomical sections occurred during the middle and late Eocene and Oligocene periods, whereas diversification within sections occurred mostly during the late Oligocene and Miocene, which is consistent with the climate warming and accompanying large distributional changes observed during these periods. These various results underline the importance of dispersal and vicariance in promoting geographic speciation and diversification in Fraxinus. Similarities in life history, reproductive and demographic attributes as well as geographical distribution patterns suggest that many other temperate trees should exhibit similar speciation patterns. On the other hand, the observed parallel evolution and reversions in floral morphology would imply a major influence of environmental pressure. The phylogeny obtained and its biogeographical implications should facilitate future studies on the evolution of complex adaptive characters, such as habitat preference, and their possible roles in promoting divergent evolution in trees.

Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting

Non-coding RNAs (ncRNAs) have emerged as versatile master regulator of biological functions in recent years. MicroRNAs (miRNAs) are small endogenous ncRNAs of 18–24 nucleotides in length that originates from long self-complementary precursors. Besides their direct involvement in developmental processes, plant miRNAs play key roles in gene regulatory networks and varied biological processes. Alternatively, long ncRNAs (lncRNAs) are a large and diverse class of transcribed ncRNAs whose length exceed that of 200 nucleotides. Plant lncRNAs are transcribed by different RNA polymerases, showing diverse structural features. Plant lncRNAs also are important regulators of gene expression in diverse biological processes. There has been a breakthrough in the technology of genome editing, the CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) technology, in the last decade. CRISPR loci are transcribed into ncRNA and eventually form a functional complex with Cas9 and further guide the complex to cleave complementary invading DNA. The CRISPR-Cas technology has been successfully applied in model plants such as Arabidopsis and tobacco and important crops like wheat, maize, and rice. However, all these studies are focused on protein coding genes. Information about targeting non-coding genes is scarce. Hitherto, the CRISPR-Cas technology has been exclusively used in vertebrate systems to engineer miRNA/lncRNAs, but it is still relatively unexplored in plants. While briefing miRNAs, lncRNAs and applications of the CRISPR-Cas technology in human and animals, this review essentially elaborates several strategies to overcome the challenges of applying the CRISPR-Cas technology in editing ncRNAs in plants and the future perspective of this field.

Abiotic Stress Induced Epigenetic Modifications in Plants: How Much Do We Know?

Epigenetics has evolved rapidly over the last two decades as a contemporary field of biology. In present day, it represents the heritable mitotic or even meiotic genetic change which does not alter the DNA sequence. Plants are considered as the masters of epigenetic regulation since they have the capability of rapid and reversible alteration of their epigenetic state and also maintaining a stable “memory” of it. Plants being sessile in nature are exposed to adverse environmental conditions which hampers their growth, development, productivity, and survival. They have developed intricate mechanisms at molecular level to withstand such stressful situations. Recent studies have documented the epigenetic control on stress-responsive mechanisms in response to various abiotic stresses. Several epigenetic mechanisms identified so far involve DNA methylation, histone modifications (acetylation, methylation, phosphorylation, ubiquitination, biotinylation, and sumoylation), chromatin remodeling, and small RNA (miRNA and siRNA) directed DNA methylation. Plants make wide use of DNA methylation as an epigenetic mark and undergo histone modifications to carry out transcriptional as well as posttranscriptional gene silencing programs. In this chapter, we have recapitulated the historical overview of the field of epigenetics followed by the various epigenetic mechanisms and lastly reviewed the studies related to various abiotic stress responses to understand the role of different epigenetic mechanisms in different plant species.

Molecular modeling of protein–protein interaction to decipher the structural mechanism of nonhost resistance in rice

Nonhost resistance (NHR) is the most common and durable form of plant resistance to disease-causing organisms. A successful example of NHR is the cloning of a maize R gene Rxo1 in rice and validating its function in conferring bacterial streak resistance in transgenic rice lines. In order to understand the structural mechanism of NHR in rice, we built the model of the protein–protein interaction between the encoded Rxo1 (RXO1) and AvrRXO1 (avirulence protein of rice pathogen, Xanthomonas oryzae pv. oryzicola). Interestingly, although a RXO1 homolog in rice (RHR) is present, it does not interact with AvrRXO1 in nature. We have confirmed that the specificity of RXO1–AvrRXO1 interaction originates from the structured leucine rich repeat (LRR) domain of RXO1, facilitating the recognition process, while the absence of such ordered LRR region makes RHR unfavorable to recognize AvrRXO1. We postulate that the RXO1–AvrRXO1 complex formation is a three step process where electrostatic interactions, shape complementarity and short-range interactions play an important role. The presence of the structural and physicochemical properties essential for the protein–protein recognition process empowers RXO1 to mediate NHR, which the host protein RHR lacks and consequently loses its specificity to bind with AvrRXO1. To the best of our knowledge, this is the first report on the understanding of NHR in rice from the structural perspective of protein–protein interaction.

Genome-wide identification of miRNAs and lncRNAs in Cajanus cajan

BackgroundNon-coding RNAs (ncRNAs) are important players in the post transcriptional regulation of gene expression (PTGR). On one hand, microRNAs (miRNAs) are an abundant class of small ncRNAs (~22nt long) that negatively regulate gene expression at the levels of messenger RNAs stability and translation inhibition, on the other hand, long ncRNAs (lncRNAs) are a large and diverse class of transcribed non-protein coding RNA molecules (> 200nt) that play both up-regulatory as well as down-regulatory roles at the transcriptional level. Cajanus cajan, a leguminosae pulse crop grown in tropical and subtropical areas of the world, is a source of high value protein to vegetarians or very poor populations globally. Hence, genome-wide identification of miRNAs and lncRNAs in C. cajan is extremely important to understand their role in PTGR with a possible implication to generate improve variety of crops.ResultsWe have identified 616 mature miRNAs in C. cajan belonging to 118 families, of which 578 are novel and not reported in MirBase21. A total of 1373 target sequences were identified for 180 miRNAs. Of these, 298 targets were characterized at the protein level. Besides, we have also predicted 3919 lncRNAs. Additionally, we have identified 87 of the predicted lncRNAs to be targeted by 66 miRNAs.ConclusionsmiRNA and lncRNAs in plants are known to control a variety of traits including yield, quality and stress tolerance. Owing to its agricultural importance and medicinal value, the identified miRNA, lncRNA and their targets in C. cajan may be useful for genome editing to improve better quality crop. A thorough understanding of ncRNA-based cellular regulatory networks will aid in the improvement of C. cajan agricultural traits.

Elucidating the functional role of predicted miRNAs in post-transcriptional gene regulation along with symbiosis in Medicago truncatula

Non-coding RNAs (ncRNAs) are found to be important regulator of gene expression because of their ability to modulate post-transcriptional processes. microRNAs are small ncRNAs which inhibit translational and post-transcriptional processes whereas long ncRNAs are found to regulate both transcriptional and post-transcriptional gene expression. Medicago truncatula is a well-known model plant for studying legume biology and is also used as a forage crop. In spite of its importance in nitrogen fixation and soil fertility improvement, little information is available about Medicago ncRNAs that play important role in symbiosis. To understand the role of Medicago ncRNAs in symbiosis and regulation of transcription factors, we have identified novel miRNAs and tried to establish an interaction model with their targets. 149 novel miRNAs are predicted along with their 770 target proteins. We have shown that 51 of these novel miRNAs are targeting 282 lncRNAs. We have analyzed the interactions between miRNAs and their target mRNAs as well as their targets on lncRNAs. Role of Medicago miRNAs in the regulation of various transcription factors were also elucidated. Knowledge gained from this study will have a positive impact on the nitrogen fixing ability of this important model plant, which in turn will improve the soil fertility.

Molecular and biochemical characterization of mungbean yellow mosaic India virus resistance in leguminous host Vigna mungo

Mungbean yellow mosaic India virus (MYMIV)—the causal agent of the yellow mosaic disease is responsible for severe damage of crops that are of great economic importance. In the current study, we explored the process of MYMIV infection and its natural resistance by analysing the expression of early and late viral genes at different time points in the leaves of resistant and susceptible Vigna mungo plants. Accordingly, we have periodically evaluated several biochemical parameters commonly associated with oxidative status of resistant and susceptible V. mungo plants during MYMIV infection. Our study revealed that accumulation levels of the early as well as late expressed genes of MYMIV were low and high in the resistant and susceptible plants, respectively; whereas membrane stability index (MSI) exhibited an opposite response. Moreover, a decrease in the malondialdehyde levels along with an increase in the activities/levels of different antioxidant enzymes, total phenol and H2O2 was noted during the early stages of infection in the resistant plants. Such observations argue in favour of strong defensive capability of the resistant plants in restricting the accumulation of viral RNA and generation of harmful free radicals within the studied tissue. Collectively, it appears that obstruction of viral invasion in plant cell wall, restriction in viral DNA replication, and early onset of antioxidant defense responses altogether might be responsible for MYMIV natural resistance. Such information is helpful in understanding the pathogenesis of MYMIV infection and its resistance in V. mungo and other economically important crops.