Xanthomonas oryzae pv oryzae triggers immediate transcriptomic modulations in rice

<p>Abstract</p> <p>Background</p> <p><it>Xanthomonas oryzae </it>pv <it>oryzae </it>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.</p> <p>Results</p> <p>Microarray 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.</p> <p>Conclusions</p> <p>This 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.</p

Small and large scale genomic DNA isolation protocol for chickpea (Cicer arietinum L.), suitable for molecular marker and transgenic analyses

Chickpea is an important food legume crop with high nutritional value. Lack of appropriate DNA isolation protocol is a limiting factor for any molecular studies of this crop. The present report describes a rapid and efficient protocol for small and large scale preparation of superior quality and quantity of DNA from four cultivars (JG62, WR315, C235 and ICCV89314) compared to that of earlier reports. The yield of DNA through both the methods was estimated to be approximately 80 μg per g of plant tissue. Both small and large scale preparations were essentially suitable for PCR and Southern blot hybridization analyses, which are the key steps in crop improvement programme through marker development and genetic engineering techniques.Key words: Cicer arietinum L., phenolics, restriction enzyme digestion, PCR amplification, Southern hybridization

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

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

Full Length Research Paper - Small and large scale genomic DNA isolation protocol for chickpea (Cicer arietinum L.), suitable for molecular marker and transgenic analyses

Chickpea is an important food legume crop with high nutritional value. Lack of appropriate DNA isolation protocol is a limiting factor for any molecular studies of this crop. The present report describes a rapid and efficient protocol for small and large scale preparation of superior quality and quantity of DNA from four cultivars (JG62, WR315, C235 and ICCV89314) compared to that of earlier reports. The yield of DNA through both the methods was estimated to be approximately 80 μg per g of plant tissue. Both small and large scale preparations were essentially suitable for PCR and Southern blot hybridization analyses, which are the key steps in crop improvement programme through marker development and genetic engineering techniques

<i>Fusarium oxysporum</i> f.sp. <i>ciceri</i> Race 1 Induced Redox State Alterations Are Coupled to Downstream Defense Signaling in Root Tissues of Chickpea (<i>Cicer arietinum</i> L.)

<div><p>Reactive oxygen species are known to play pivotal roles in pathogen perception, recognition and downstream defense signaling. But, how these redox alarms coordinate <i>in planta</i> into a defensive network is still intangible. Present study illustrates the role of <i>Fusarium oxysporum</i> f.sp <i>ciceri</i> 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.</p></div

Relative expression of redox signalling genes in chickpea roots in response to Foc1 induction.

<p>(A) and (B) represents relative expression profile of ROS generators and scavengers like respiratory burst oxidase homologue (RBOH), peroxidase, cationic peroxidase, iron superoxide dismutase (Fe-SOD) and glutathione s transferase TAU26 (GST-TAU26) in uninduced and induced JG62 and WR315 roots respectively; (C) and (D) represents relative expression pattern of cytochrome dependent redox signal transducers like cytochrome b561 Fe reductase, FAD linked oxidase family protein and NADH cytochrome b5 reductase in uninduced and induced JG62 and WR315 roots respectively; (E) and (F) represents relative expression profile of intracellular ROS signal transducers like NADP oxido-reductase, quinone oxido-reductase, Fe(II) oxido-reductase, F type thioredoxin and H⁺ transporting ATPase in uninduced and induced JG62 and WR315 roots respectively. Bars represent the standard errors (n = 3).</p

Confocal microscopic images showing Foc1 infection induced nuclear migration in root cells of chickpea plants.

<p>(A–C) correspond to root sections of uninduced JG 62 plant; (D–F) correspond to root sections of infected JG62 plant at 7dpi; (G–I) of infected JG62 plant at 12dpi; (J–L) correspond to root sections of infected WR315 plant at 12dpi. A, D, G, J represent fluorescent images; B, E, H, K represent DIC images; C, F, I, L represent merged images. Bars correspond to 10 µm.</p