Sumanti Gupta

Primary Metabolism of Chickpea Is the Initial Target of Wound Inducing Early Sensed Fusarium oxysporum f. sp. ciceri Race I

Background Biotrophic interaction between host and pathogen induces generation of reactive oxygen species that leads to programmed cell death of the host tissue specifically encompassing the site of infection conferring resistance to the host. However, in the present study, biotrophic relationship between Fusarium oxysporum and chickpea provided some novel insights into the classical concepts of defense signaling and disease perception where ROS (reactive oxygen species) generation followed by hypersensitive responses determined the magnitude of susceptibility or resistant potentiality of the host. Methodology/Principal Findings Microscopic observations detected wound mediated in planta pathogenic establishment and its gradual progression within the host vascular tissue. cDNA-AFLP showed differential expression of many defense responsive elements. Real time expression profiling also validated the early recognition of the wound inducing pathogen by the host. The interplay between fungus and host activated changes in primary metabolism, which generated defense signals in the form of sugar molecules for combating pathogenic encounter. Conclusions/Significance The present study showed the limitations of hypersensitive response mediated resistance, especially when foreign encounters involved the food production as well as the translocation machinery of the host. It was also predicted from the obtained results that hypersensitivity and active species generation failed to impart host defense in compatible interaction between chickpea and Fusarium. On the contrary, the defense related gene(s) played a critical role in conferring natural resistance to the resistant host. Thus, this study suggests that natural selection is the decisive factor for selecting and segregating out the suitable type of defense mechanism to be undertaken by the host without disturbing its normal metabolism, which could deviate from the known classical defense mechanisms.

Xanthomonas oryzae pv oryzae triggers immediate transcriptomic modulations in rice

BackgroundXanthomonas oryzae pv oryzae is a devastating pathogen of rice and has been extensively studied as a model pathogen of monocotyledons. Expressional studies in both the contenders have been undertaken in past to understand the molecular mechanism underlying the compatible and incompatible interactions in the pathosystem. Continuous update on database and gene annotations necessitates constant updating on the roles of the new entities as well as reinterpretation of regulations of the previous ones. Moreover the past endeavors have addressed the middle or late defense responses of the rice plant whereas in the present study an attempt has been made to investigate the early defense responses taking place immediately after inoculation.ResultsMicroarray was used to study the transcriptional modulations in eighteen days old rice seedling leaves of both susceptible and resistant genotypes one hour after inoculation. In resistant plants as compared to susceptible ones 274 genes were found to be differentially expressed. Annotations could be assigned to 112 up- and 73 down-regulated transcripts and gene interaction maps were generated for 86 transcripts. Expressional data and interaction maps were used to develop a hypothetical scheme of the molecular events taking place during early defense response. Network analysis with the differential transcripts showed up-regulation of major clusters of cell signaling proteins and transcription factors while growth and basal metabolic components were largely found to be down-regulated.ConclusionsThis study provides an understanding of the early defense signaling in rice cells. Components of the calcium and lipid signaling as well as MAPK cascade were modulated, by signals from surface receptors and cytosolic R-proteins, to arouse jasmonic acid and ethylene signaling and suppress auxin signaling through various transcription factors. Abscisic acid modulation was also evident through the expression regulation of transcription factors involved with its functions. Moreover adjustments in expression levels of components of primary as well as secondary metabolism, protein trafficking and turnout were apparent, highlighting the complexity of defense response.

A Molecular Insight into the Early Events of Chickpea (Cicer arietinum) and Fusarium oxysporum f. sp. ciceri (Race 1) Interaction Through cDNA-AFLP Analysis

Wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris is one of the most severe diseases of chickpea throughout the world. Variability of pathotypes of F. oxysporum f. sp. ciceris and breakdown of natural resistance are the main hindrances to developing resistant plants by applying resistant breeding strategies. Additionally, lack of information of potential resistant genes limits gene-transfer technology. A thorough understanding of Fusarium spp.-chickpea interaction at a cellular and molecular level is essential for isolation of potential genes involved in counteracting disease progression. Experiments were designed to trigger the pathogen-challenged disease responses in both susceptible and resistant plants and monitor the expression of stress induced genes or gene fragments at the transcript level. cDNA amplified fragment length polymorphism followed by homology search helped in differentiating and analyzing the up- and downregulated gene fragments. Several detected DNA fragments appeared to have relevance with pathogen-mediated defense. Some of the important transcript-derived fragments were homologous to genes for sucrose synthase, isoflavonoid biosynthesis, drought stress response, serine threonine kinases, cystatins, arginase, and so on. Reverse-transcriptase polymerase chain reaction performed with samples collected at 48 and 96 h postinfection confirmed a similar type of differential expression pattern. Based on these results, interacting pathways of cellular processes were generated. This study has an implication toward functional identification of genes involved in wilt resistance.

Optimization of an Efficient Protein Extraction Protocol Compatible with Two-Dimensional Electrophoresis and Mass Spectrometry from Recalcitrant Phenolic Rich Roots of Chickpea (Cicer arietinum L.)

Two-dimensional electrophoresis and mass spectrometry are undoubtedly two essential tools popularly used in proteomic analyses. Utilization of these techniques however largely depends on efficient and optimized sample preparation, regarded as one of the most crucial steps for recovering maximum amount of reliable information. The present study highlights the optimization of an effective and efficient protocol, capable of extraction of root proteins from recalcitrant phenolic rich tissues of chickpea. The widely applicable TCA-acetone and phenol-based methods have been comparatively evaluated, amongst which the latter appeared to be better suited for the sample. The phenol extraction-based method further complemented with sodium dodecyl sulphate (SDS) and pulsatory treatments proved to be the most suitable method represented by greatest spot number, good resolution, and spot intensities. All the randomly selected spots showed successful identification when subjected to further downstream MALDI-TOF and MS/MS analyses. Hence, the information obtained collectively proposes the present protein extraction protocol to be an effective one that could be applicable for recalcitrant leguminous root samples.

Fusarium oxysporum f.sp. ciceri race 1 induced redox state alterations are coupled to downstream defense signaling in root tissues of chickpea (Cicer arietinum L.).

Reactive oxygen species are known to play pivotal roles in pathogen perception, recognition and downstream defense signaling. But, how these redox alarms coordinate in planta into a defensive network is still intangible. Present study illustrates the role of Fusarium oxysporum f.sp ciceri Race1 (Foc1) induced redox responsive transcripts in regulating downstream defense signaling in chickpea. Confocal microscopic studies highlighted pathogen invasion and colonization accompanied by tissue damage and deposition of callose degraded products at the xylem vessels of infected roots of chickpea plants. Such depositions led to the clogging of xylem vessels in compatible hosts while the resistant plants were devoid of such obstructions. Lipid peroxidation assays also indicated fungal induced membrane injury. Cell shrinkage and gradual nuclear adpression appeared as interesting features marking fungal ingress. Quantitative real time polymerase chain reaction exhibited differential expression patterns of redox regulators, cellular transporters and transcription factors during Foc1 progression. Network analysis showed redox regulators, cellular transporters and transcription factors to coordinate into a well orchestrated defensive network with sugars acting as internal signal modulators. Respiratory burst oxidase homologue, cationic peroxidase, vacuolar sorting receptor, polyol transporter, sucrose synthase, and zinc finger domain containing transcription factor appeared as key molecular candidates controlling important hubs of the defense network. Functional characterization of these hub controllers may prove to be promising in understanding chickpea–Foc1 interaction and developing the case study as a model for looking into the complexities of wilt diseases of other important crop legumes.

Analysis of root proteome unravels differential molecular responses during compatible and incompatible interaction between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp. ciceri Race1 (Foc1)

BackgroundVascular wilt caused by Fusarium oxysporum f. sp. ciceri Race 1 (Foc1) is a serious disease of chickpea (Cicer arietinum L.) accounting for approximately 10-15% annual crop loss. The fungus invades the plant via roots, colonizes the xylem vessels and prevents the upward translocation of water and nutrients, finally resulting in wilting of the entire plant. Although comparative transcriptomic profiling have highlighted some important signaling molecules, but proteomic studies involving chickpea-Foc1 are limited. The present study focuses on comparative root proteomics of susceptible (JG62) and resistant (WR315) chickpea genotypes infected with Foc1, to understand the mechanistic basis of susceptibility and/or resistance.ResultsThe differential and unique proteins of both genotypes were identified at 48 h, 72 h, and 96 h post Foc1 inoculation. 2D PAGE analyses followed by MALDI-TOF MS and MS/MS identified 100 differentially (>1.5 fold<, p < 0.05) or uniquely expressed proteins. These proteins were further categorized into 10 functional classes and grouped into GO (gene ontology) categories. Network analyses of identified proteins revealed intra and inter relationship of these proteins with their neighbors as well as their association with different defense signaling pathways. qRT-PCR analyses were performed to correlate the mRNA and protein levels of some proteins of representative classes.ConclusionsThe differential and unique proteins identified indicate their involvement in early defense signaling of the host. Comparative analyses of expression profiles of obtained proteins suggest that albeit some common components participate in early defense signaling in both susceptible and resistant genotypes, but their roles and regulation differ in case of compatible and/or incompatible interactions. Thus, functional characterization of identified PR proteins (PR1, BGL2, TLP), Trypsin protease inhibitor, ABA responsive protein, cysteine protease, protein disulphide isomerase, ripening related protein and albumins are expected to serve as important molecular components for biotechnological application and development of sustainable resistance against Foc1.

Binding of insecticidal lectin Colocasia esculenta tuber agglutinin (CEA) to midgut receptors of Bemisia tabaci and Lipaphis erysimi provides clues to its insecticidal potential.

The insecticidal potential of Galanthus nivalis agglutinin‐related lectins against hemipterans has been experimentally proven. However, the basis behind the toxicity of these lectins against hemipterans remains elusive. The present study elucidates the molecular basis behind insecticidal efficacy of Colocasia esculenta tuber agglutinin (CEA) against Bemisia tabaci and Lipaphis erysimi. Confocal microscopic analyses highlighted the binding of 25 kDa stable homodimeric lectin to insect midgut. Ligand blots followed by LC MS/MS analyses identified binding partners of CEA as vacuolar ATP synthase and sarcoplasmic endoplasmic reticulum type Ca2+ ATPase from B. tabaci, and ATP synthase, heat shock protein 70 and clathrin heavy chain assembly protein from L. erysimi. Internalization of CEA into hemolymph was confirmed by Western blotting. Glycoprotein nature of the receptors was identified through glycospecific staining. Deglycosylation assay indicated the interaction of CEA with its receptors to be probably glycan mediated. Surface plasmon resonance analysis revealed the interaction kinetics between ATP synthase of B. tabaci with CEA. Pathway prediction study based on Drosophila homologs suggested the interaction of CEA with insect receptors that probably led to disruption of cellular processes causing growth retardation and loss of fecundity of target insects. Thus, the present findings strengthen our current understanding of the entomotoxic potentiality of CEA, which will facilitate its future biotechnological applications.

Understanding the molecular defence responses of host during chickpea-Fusarium interplay: where do we stand?

Fusarium oxysporum is known to cause vascular wilt and root rot of many important plants. Although extensive studies have been reported for the model plant Arabidopsis thaliana (L.) Heynh., the question of whether those experimental interpretations are extendable to other crop species requires experimentation. Chickpea is the most important crop legume of Indian subcontinent and ranks third in the world list of important legumes. However, productivity of this crop is severely curtailed by vascular wilt caused by Fusarium oxysporum f. sp. ciceri. Based on earlier reports, the present review discusses about the external manifestations of the disease, in planta fungal progression and establishment, and the molecular responses of chickpea that occur during Fusarium oxysporum f. sp. ciceri Race 1(Foc1) interaction. Foc1, known to enter the roots through the breaches of tap root, colonise the xylem vessels and block upward translocation of essential solutes causing wilt in compatible hosts. In contrast, pathogen invasion is readily perceived by the resistant host, which activates defence signalling cascades that are directed towards protecting its primary metabolism from the harmful consequences of pathogenic mayhem. Hence, understanding the dynamic complexities of chickpea-Foc1 interplay is prerequisite to providing sustainable solutions in wilt management programs.

In search of decoy/guardee to R genes Deciphering the role of sugars in defense against Fusarium wilt in chickpea

Plant responses are coordinately controlled by both external and internal signals. Apt perception of pathogen attack and its appropriate conversion to internal signals ultimately determine the outcome of innate immunity. The present review predicts the involvement of unconventional ‘Guard/Decoy Model’ in chickpea-Fusarium encounter. Rapid alkalinization factor is predicted to act as initial ‘Gatekeeper decoy’ counteracting fungal entry. Phospholipases and cystatins probably function as ‘Guardees’ being shielded by R gene(s). Serine Threonine Kinases decodes external pathogenic signals to in planta defense alarms. 14.3.3 provides clues to the wilt mechanism. The versatile sugars serve as signal generators and transmitters maintaining intra and inter cellular connectivity during stress.

Transcriptomic dissection reveals wide spread differential expression in chickpea during early time points of Fusarium oxysporum f. sp. ciceri Race 1 attack

Plants’ reaction to underground microorganisms is complex as sessile nature of plants compels them to prioritize their responses to diverse microorganisms both pathogenic and symbiotic. Roots of important crops are directly exposed to diverse microorganisms, but investigations involving root pathogens are significantly less. Thus, more studies involving root pathogens and their target crops are necessitated to enrich the understanding of underground interactions. Present study reported the molecular complexities in chickpea during Fusarium oxysporum f. sp. ciceri Race 1 (Foc1) infection. Transcriptomic dissections using RNA-seq showed significantly differential expression of molecular transcripts between infected and control plants of both susceptible and resistant genotypes. Radar plot analyses showed maximum expressional undulations after infection in both susceptible and resistant plants. Gene ontology and functional clustering showed large number of transcripts controlling basic metabolism of plants. Network analyses demonstrated defense components like peptidyl cis/trans isomerase, MAP kinase, beta 1,3 glucanase, serine threonine kinase, patatin like protein, lactolylglutathione lyase, coproporphyrinogen III oxidase, sulfotransferases; reactive oxygen species regulating components like respiratory burst oxidase, superoxide dismutases, cytochrome b5 reductase, glutathione reductase, thioredoxin reductase, ATPase; metabolism regulating components, myo inositol phosphate, carboxylate synthase; transport related gamma tonoplast intrinsic protein, and structural component, ubiquitins to serve as important nodals of defense signaling network. These nodal molecules probably served as hub controllers of defense signaling. Functional characterization of these hub molecules would not only help in developing better understanding of chickpea-Foc1 interaction but also place them as promising candidates for resistance management programs against vascular wilt of legumes.