Papers by Muthappa Senthil-kumar
Frontiers in plant science, 2015
In field conditions, plants are often simultaneously exposed to multiple biotic and abiotic stres... more In field conditions, plants are often simultaneously exposed to multiple biotic and abiotic stresses resulting in substantial yield loss. Plants have evolved various physiological and molecular adaptations to protect themselves under stress combinations. Emerging evidences suggest that plant responses to a combination of stresses are unique from individual stress responses. In addition, plants exhibit shared responses which are common to individual stresses and stress combination. In this review, we provide an update on the current understanding of both unique and shared responses. Specific focus of this review is on heat-drought stress as a major abiotic stress combination and, drought-pathogen and heat-pathogen as examples of abiotic-biotic stress combinations. We also comprehend the current understanding of molecular mechanisms of cross talk in relation to shared and unique molecular responses for plant survival under stress combinations. Thus, the knowledge of shared responses o...
Frontiers in plant science, 2015
Nutrients are indispensable elements required for the growth of all living organisms including pl... more Nutrients are indispensable elements required for the growth of all living organisms including plants and pathogens. Phyllosphere, rhizosphere, apoplast, phloem, xylem, and cell organelles are the nutrient niches in plants that are the target of bacterial pathogens. Depending upon nutrients availability, the pathogen adapts various acquisition strategies and inhabits the specific niche. In this review, we discuss the nutrient composition of different niches in plants, the mechanisms involved in the recognition of nutrient niche and the sophisticated strategies used by the bacterial pathogens for acquiring nutrients. We provide insight into various nutrient acquisition strategies used by necrotrophic, biotrophic, and hemibiotrophic bacteria. Specifically we discuss both modulation of bacterial machinery and manipulation of host machinery. In addition, we highlight the current status of our understanding about the nutrient acquisition strategies used by bacterial pathogens, namely tar...
Frontiers in Plant Science, 2015
Qamar A, Mysore KS and Senthil-Kumar M (2015) Role of proline and pyrroline-5-carboxylate metabol... more Qamar A, Mysore KS and Senthil-Kumar M (2015) Role of proline and pyrroline-5-carboxylate metabolism in plant defense against invading pathogens. Front. Plant Sci. 6:503. Pyrroline-5-carboxylate (P5C) is an intermediate product of both proline biosynthesis and catabolism. Recent evidences indicate that proline-P5C metabolism is tightly regulated in plants, especially during pathogen infection and abiotic stress. However, role of P5C and its metabolism in plants has not yet been fully understood. Studies indicate that P5C synthesized in mitochondria has a role in both resistance (R)-gene-mediated and non-host resistance against invading pathogens. Proline dehydrogenase and delta-ornithine amino transferase-encoding genes, both involved in P5C synthesis in mitochondria are implicated in defense response of Nicotiana benthamiana and Arabidopsis thaliana against bacterial pathogens. Such defense response is proposed to involve salicylic acid-dependent pathway, reactive oxygen species (ROS) and hypersensitive response (HR)-associated cell death. Recently HR, a form of programmed cell death (PCD), has been proposed to be induced by changes in mitochondrial P5C synthesis or the increase in P5C levels per se in plants inoculated with either a host pathogen carrying suitable avirulent (Avr) gene or a non-host pathogen. Consistently, A. thaliana mutant plants deficient in P5C catabolism showed HR like cell death when grown in external P5C or proline supplemented medium. Similarly, yeast and plant cells under oxidative stress were shown to increase ROS production and PCD due to increase in P5C levels. Similar mechanism has also been reported as one of the triggers for apoptosis in mammalian cells. This review critically analyzes results from various studies and enumerates the pathways for regulation of P5C levels in the plant cell, especially in mitochondria, during pathogen infection. Further, mechanisms regulating P5C-mediated defense responses, namely HR are outlined. This review also provides new insights into the differential role of proline-P5C metabolism in plants exposed to pathogen infection.
Journal of Visualized Experiments, 2013
Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense... more Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana. In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.
Frontiers in Plant Science, 2014
Plants are constantly exposed to microorganisms in the environment and, as a result, have evolved... more Plants are constantly exposed to microorganisms in the environment and, as a result, have evolved intricate mechanisms to recognize and defend themselves against potential pathogens. One of these responses is the downregulation of photosynthesis and other processes associated with primary metabolism that are essential for plant growth. It has been suggested that the energy saved by downregulation of primary metabolism is diverted and used for defense responses. However, several studies have shown that upregulation of primary metabolism also occurs during plant-pathogen interactions. We propose that upregulation of primary metabolism modulates signal transduction cascades that lead to plant defense responses. In support of this thought, we here compile evidence from the literature to show that upon exposure to pathogens or elicitors, plants induce several genes associated with primary metabolic pathways, such as those involved in the synthesis or degradation of carbohydrates, amino acids and lipids. In addition, genetic studies have confirmed the involvement of these metabolic pathways in plant defense responses. This review provides a new perspective highlighting the relevance of primary metabolism in regulating plant defense against pathogens with the hope to stimulate further research in this area.
In nature, plants are simultaneously exposed to a combination of biotic and abiotic stresses that... more In nature, plants are simultaneously exposed to a combination of biotic and abiotic stresses that limit crop yields. Only recently, researchers have started understanding the molecular basis of combined biotic and abiotic stress interactions. Evidences suggest that under combined stress plants exhibit tailored physiological and molecular responses, in addition to several shared responses as part of their stress tolerance strategy. These tailored responses are suggested to occur only in plants exposed to simultaneous stresses and this information cannot be inferred from individual stress studies. In this review article, we provide update on the responses of plants to simultaneous biotic and abiotic stresses, in particular drought and pathogen. Simultaneous occurrence of drought and pathogen during plant growth provokes complex pathways controlled by different signaling events resulting in positive or negative impact of one stress over the other. Here, we summarize the effect of combined drought and pathogen infection on plants and highlight the tailored strategies adapted by plants. Besides, we enumerate the evidences from pathogen derived elicitors and ABA response studies for understanding simultaneous drought and pathogen tolerance.
BMC Plant Biology, 2013
Background: Understanding the function of a particular gene under various stresses is important f... more Background: Understanding the function of a particular gene under various stresses is important for engineering plants for broad-spectrum stress tolerance. Although virus-induced gene silencing (VIGS) has been used to characterize genes involved in abiotic stress tolerance, currently available gene silencing and stress imposition methodology at the whole plant level is not suitable for high-throughput functional analyses of genes. This demands a robust and reliable methodology for characterizing genes involved in abiotic and multi-stress tolerance. Results: Our methodology employs VIGS-based gene silencing in leaf disks combined with simple stress imposition and effect quantification methodologies for easy and faster characterization of genes involved in abiotic and multi-stress tolerance. By subjecting leaf disks from gene-silenced plants to various abiotic stresses and inoculating silenced plants with various pathogens, we show the involvement of several genes for multi-stress tolerance. In addition, we demonstrate that VIGS can be used to characterize genes involved in thermotolerance. Our results also showed the functional relevance of NtEDS1 in abiotic stress, NbRBX1 and NbCTR1 in oxidative stress; NtRAR1 and NtNPR1 in salinity stress; NbSOS1 and NbHSP101 in biotic stress; and NtEDS1, NbETR1, NbWRKY2 and NbMYC2 in thermotolerance.
Methods in Molecular Biology, 2010
Agroinoculation, first developed as a simple tool to study plant-virus interactions, is a popular... more Agroinoculation, first developed as a simple tool to study plant-virus interactions, is a popular method of choice for functional gene analysis of viral genomes. With the explosive growth of genomic information and the development of advanced vectors to dissect plant gene function, this reliable method of viral gene delivery in plants, has been recruited and morphed into a technique popularly known as agroinfiltration. This technique was developed to examine the effects of transient gene expression, with applications ranging from studies of plant-pathogen interactions, abiotic stresses, a variety of transient expression assays to study protein localization, and protein-protein interactions. We present a brief overview of literature which document both these applications, and then provide simple agroinoculation and agroinfiltration methods being used in our laboratory for functional gene analysis, as well as for fast-forward and reverse genetic screens using virus-induced gene silencing (VIGS).
Frontiers in Plant Science, 2014
Over the last decade, VIGS has been successfully used as both a forward and reverse genetics tech... more Over the last decade, VIGS has been successfully used as both a forward and reverse genetics technique for gene function analysis in various model plants, as well as crop plants. With the increased identification of differentially expressed genes under various abiotic stresses through high-throughput transcript profiling, the application of VIGS is expected to be important in the future for functional characterization of a large number of genes. In the recent past, VIGS was proven to be an elegant tool for functional characterization of genes associated with abiotic stress responses. In this review, we provide an overview of how VIGS is used in different crop species to characterize genes associated with drought-, salt-, oxidative-and nutrient-deficiency-stresses. We describe the examples from studies where abiotic stress related genes are characterized using VIGS. In addition, we describe the major advantages of VIGS over other currently available functional genomics tools. We also summarize the recent improvements, limitations and future prospects of using VIGS as a tool for studying plant responses to abiotic stresses.
Annual Review of Phytopathology, 2013
Nonhost resistance is a broad-spectrum plant defense that provides immunity to all members of a p... more Nonhost resistance is a broad-spectrum plant defense that provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Upon landing on the surface of a nonhost plant species, a potential bacterial pathogen initially encounters preformed and, later, induced plant defenses. One of the initial defense responses from the plant is pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Nonhost plants also have mechanisms to detect nonhost-pathogen effectors and can trigger a defense response referred to as effector-triggered immunity (ETI). This nonhost resistance response often results in a hypersensitive response (HR) at the infection site. This review provides an overview of these plant defense strategies. We enumerate plant genes that impart nonhost resistance and the bacterial counter-defense strategies. In addition, prospects for application of nonhost resistance to achieve broad-spectrum and durable resistance in crop plants are also discussed.
International Journal of Molecular Sciences, 2013
Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a... more Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a priming response has not been widely studied. In particular, little is known about enhanced defense capacity of drought stress acclimated plants to fungal and bacterial pathogens. Here we show that prior drought acclimation in Nicotiana benthamiana plants imparts tolerance to necrotrophic fungus, Sclerotinia sclerotiorum, and also to hemi-biotrophic bacterial pathogen, Pseudomonas syringae pv. tabaci. S. sclerotiorum inoculation on N. benthamiana plants acclimated with drought stress lead to less disease-induced cell death compared to non-acclimated plants. Furthermore, inoculation of P. syringae pv. tabaci on N. benthamiana plants acclimated to moderate drought stress showed reduced disease symptoms. The levels of reactive oxygen species (ROS) in drought acclimated plants were highly correlated with disease resistance. Further, in planta growth of GFPuv expressing P. syringae pv. tabaci on plants pre-treated with methyl viologen showed complete inhibition of bacterial growth. Taken together, these experimental results suggested a role for ROS generated during drought acclimation in imparting tolerance against S. sclerotiorum and P. syringae pv. tabaci. We speculate that the generation of ROS during drought acclimation primed a defense response in plants that subsequently caused the tolerance against the pathogens tested.
PLoS ONE, 2014
Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the ... more Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase (mtlD), were developed through Agrobacterium tumefaciens-mediated genetic transformation. mtlD gene integration in the putative transgenic plants was confirmed by Southern blot. Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet.
Methods in Molecular Biology, 2011
RNA interference (RNAi), mediated by short interfering RNAs (siRNAs), is one of the widely used f... more RNA interference (RNAi), mediated by short interfering RNAs (siRNAs), is one of the widely used functional genomics method for suppressing the gene expression in plants. Initially, gene silencing by RNAi mechanism was believed to be specific requiring sequence homology between siRNA and target mRNA. However, several recent reports have showed that non-specific effects often referred as off-target gene silencing can occur during RNAi. This unintended gene silencing can lead to false conclusions in RNAi experiments that are aimed to study the functional role of a particular target gene in plants. This especially is a major problem in large-scale RNAi-based screens aiming for gene discovery. Hence, understanding the off-target effects is crucial for minimizing such effects to better conclude gene function analyzed by RNAi. We discuss here potential problems of off-target gene silencing and focus on possibilities that favor this effect during post-transcriptional gene silencing. Suggestions to overcome the off-target effects during RNAi studies are also presented. We believe that information available in present-day plant science literature about specificity of siRNA actions is inadequate. In-depth systematic studies to understand their molecular basis are necessary to enable improved design of more specific RNAi vectors. In the meantime, gene function and phenotype results from present-day RNAi studies need to be interpreted with caution.
Trends in Plant Science, 2011
Virus-induced gene silencing (VIGS) is an efficient tool for gene function studies. It has been u... more Virus-induced gene silencing (VIGS) is an efficient tool for gene function studies. It has been used to perform both forward and reverse genetics to identify plant genes involved in several plant processes. However, this technology has not yet been used to its full potential. This can be attributed to several of its limitations such as inability to silence genes during seed germination and the non-stable nature of silencing. However, several recent studies have shown that these limitations can now be overcome. In this review, we will discuss the limitations of VIGS and suitable solutions. In addition, we also describe the recent improvements and future prospects of using VIGS in plant biology.
PLoS ONE, 2012
NAC (NAM, ATAF1-2, and CUC2) proteins constitute one of the largest families of plant-specific tr... more NAC (NAM, ATAF1-2, and CUC2) proteins constitute one of the largest families of plant-specific transcription factors and have been shown to be involved in diverse plant processes including plant growth, development, and stress-tolerance. In this study, a stress-responsive NAC gene, EcNAC1, was isolated from the subtracted stress cDNA library generated from a drought adapted crop, finger millet, and characterized for its role in stress-tolerance. The expression analysis showed that EcNAC1 was highly induced during water-deficit and salt stress. EcNAC1 shares high amino acid similarity with rice genes that have been phylogenetically classified into stress-related NAC genes. Our results demonstrated that tobacco transgenic plants expressing EcNAC1 exhibit tolerance to various abiotic stresses like simulated osmotic stress, by polyethylene glycol (PEG) and mannitol, and salinity stress. The transgenic plants also showed enhanced tolerance to methyl-viologen (MV) induced oxidative stress. Reduced levels of reactive oxygen species (ROS) and ROS-induced damage were noticed in pot grown transgenic lines under water-deficit and natural high light conditions. Root growth under stress and recovery growth after stress alleviation was more in transgenic plants. Many stress-responsive genes were found to be up-regulated in transgenic lines expressing EcNAC1. Our results suggest that EcNAC1 overexpression confers tolerance against abiotic stress in susceptible species, tobacco.
Planta, 2007
Stress tolerance is a multigenic character and there are many stress responsive genes, which are ... more Stress tolerance is a multigenic character and there are many stress responsive genes, which are stress speciWc. Although many of these have been cloned, their functional signiWcance remains fragmentary. Hence it is important to identify the relevant stress genes involved in altering the metabolism for adaptation. Overexpression is one of the several approaches and Chlamydomonas is a suitable system to study the functional relevance of stress genes. Stress responses can only be assessed on prior exposure to sublethal induction stress. In this study the acclimation response of Chlamydomonas was assessed for diVerent abiotic stresses using physiological screens like chlorophyll stability, membrane damage, cell viability, accumulation of free radicals, survival and recovery growth. We demonstrate that Chlamydomonas responds to diverse stresses and is a potential system to study the relevance of stress genes. The relevance of choline oxidase A (codA), a key enzyme in glycinebetaine biosynthesis, was examined by developing transformants expressing codA gene from Arthrobacter globiformis. Southern positive transformants showed enhanced accumulation of glycinebetaine. The transformants also showed enhanced growth under salinity, high light coupled with methylviologen-induced oxidative stress, high temperature and cold stress. However the transgenics were not tolerant to PEG-mediated simulated osmotic stress, LiCl, menadione and UV stress. Increased cell survival and decreased chlorophyll degradation in transformants under acclimated conditions further con-Wrmed the relevance of codA in imparting stress tolerance. Our results indicated that the relevance of stress responsive genes can be eYciently validated for diverse abiotic stresses using Chlamydomonas system.
Planta, 2007
Determining the functional role of genes that are differentially regulated during a stress respon... more Determining the functional role of genes that are differentially regulated during a stress response is challenging. In this study, few water deficitinduced genes from peanut were characterized in Nicotiana benthamiana using virus-induced gene silencing (VIGS) and their relevance for stress adaptation was validated. Twenty-five cDNA clones from peanut water deficit stress-induced cDNA library that had more than 50% nucleotide similarity with N. benthamiana or tomato homologs were selected. VIGS in peanut is not yet feasible and therefore we characterized these 25 genes in N. benthamiana. Increased membrane damage was seen under water deficit stress in most of the silenced plants signifying that many of these stress-induced genes are important to confer drought tolerance. Among the genes tested, silencing by homolog of flavonol 3-O-glucosyltransferase (F3OGT), homolog of alcohol dehydrogenase, homologous to salt inducible protein, and homolog of heat shock protein 70 showed more visible wilting symptoms compared with the control plants during water deficit stress. Interestingly, down-regulation of two genes, homologous to aspartic proteinase 2, and homolog of Jumonji class of transcription factor showed relative drought tolerant phenotypes. F3OGT silenced plants showed more wilting symptoms, membrane damage and chlorophyll degradation than any other silenced plants during water deficit. Our results demonstrate that VIGS approach can be used to characterize and assess the functional relevance of water deficit stress-induced cDNAs in a heterologous species.
Plant, Cell & Environment, 2012
Non-host disease resistance involves the production of hypersensitive response (HR), a programmed... more Non-host disease resistance involves the production of hypersensitive response (HR), a programmed cell death (PCD) that occurs at the site of pathogen infection. Plant mitochondrial reactive oxygen species (ROS) production and red-ox changes play a major role in regulating such cell death. Proline catabolism reactions, especially pyrroline-5carboxylate (P5C) accumulation, are known to produce ROS and contribute to cell death. Here we studied important genes related to proline synthesis and catabolism in the defence against host and non-host strains of Pseudomonas syringae in Nicotiana benthamiana and Arabidopsis. Our results show that ornithine delta-aminotransferase (dOAT) and proline dehydrogenases (ProDH1 and ProDH2) are involved in the defence against non-host pathogens. Silencing of these genes in N. benthamiana delayed occurrence of HR and favoured non-host pathogen growth. Arabidopsis mutants for these genes compromised non-host resistance and showed a decrease in non-host pathogen-induced ROS. Some of the genes involved in proline metabolism were also induced by a pathogen-carrying avirulence gene, indicating that proline metabolism is influenced during effectortriggered immunity (ETI). Our results demonstrate that dOAT and ProDH enzyme-mediated steps produce ROS in mitochondria and regulate non-host HR, thus contributing to non-host resistance in plants.
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Papers by Muthappa Senthil-kumar