kishor ingole

Ustilago maydis is a biotrophic phytopathogenic fungus that causes corn smut disease. As a well-established model system, U. maydis is genetically fully accessible with large omics datasets available and subject to various biological questions ranging from DNA-repair, RNA-transport, and protein secretion to disease biology. For many genetic approaches, tight control of transgene regulation is important. Here we established an optimised version of the Tetracycline-ON (TetON) system for U. maydis. We demonstrate the Tetracycline concentration-dependent expression of fluorescent protein transgenes and the system’s suitability for the induced expression of the toxic protein BCL2 Associated X-1 (Bax1). The Golden Gate compatible vector system contains a native minimal promoter from the mating factor a-1 encoding gene, mfa with ten copies of the tet-regulated operator (tetO) and a codon optimised Tet-repressor (tetR*) which is translationally fused to the native transcriptional corepresso...

Pseudomonas syringae-secreted HopA1 effectors are important determinants in host range expansion and increased pathogenicity. Their recent acquisitions via horizontal gene transfer in several non-pathogenic Pseudomonas strains worldwide have caused alarming increase in their virulence capabilities. In Arabidopsis thaliana, RESISTANCE TO PSEUDOMONAS SYRINGAE 6 (RPS6) gene confers effector-triggered immunity (ETI) against HopA1pss derived from P. syringae pv. syringae strain 61. Surprisingly, a closely related HopA1pst from the tomato pathovar evades immune detection. These responsive differences in planta between the two HopA1s represents a unique system to study pathogen adaptation skills and host-jumps. However, molecular understanding of HopA1′s contribution to overall virulence remain undeciphered. Here, we show that immune-suppressive functions of HopA1pst are more potent than HopA1pss. In the resistance-compromised ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) null-mutant, transcrip...

The attachment of SMALL UBIQUITIN-LIKE MODIFIER (SUMO) to target proteins regulates a plethora of cellular processes across eukaryotes. In Arabidopsis thaliana, mutants with abnormal SUMO1/2 conjugate levels display a dwarf stature, autoimmunity, and altered stress responses to adverse environmental conditions. Since the SUMO pathway is known to autoregulate its biochemical activity (via allosteric interactions), we assessed whether the emergence of additional SUMO paralogs in Arabidopsis has introduced the capacity of self-regulation by means of isoform diversification in this model plant. By studying the plant defense responses elicited by the bacterial pathogen Pseudomonas syringae pv. tomato, we provide genetic evidence that SUM3, a divergent paralog, acts downstream of the two main SUMO paralogues, SUM1/2. The expression of SUM3 apparently buffers or suppresses the function of SUM1/2 by controlling the timing and amplitude of the immune response. Moreover, SUM1 and SUM2 work ad...

Steady-state SUMOylome of a plant is adjusted locally during developmental transitions and more globally during stress exposures. We recently reported that basal immunity in Arabidopsis thaliana against Pseudomonas syringae pv tomato strain DC3000 (PstDC3000) is associated with strong enhancements in the net SUMOylome. Transcriptional upregulations of SUMO conjugases, suppression of protease, and increased SUMO translations accounted for this enhanced SUMOylation. Antagonistic roles of SUMO1/2 and SUMO3 isoforms further fine-tuned the SUMOylome adjustments, thus impacting defense amplitudes and immune outcomes. Loss of function of SUPPRESSOR OF rps4-RLD1 (SRFR1), a previously reported negative regulator of basal defenses, also caused constitutive increments in global SUMO-conjugates through similar modes. These suggest that SRFR1 plays a pivotal role in maintenance of SUMOylation homeostasis and its dynamic changes during immune elicitations. Here, we demonstrate that SRFR1 degradat...

Targeted protein degradation is essential for physiological development and adaptation to stress. Mammalian INOSITOL PENTAKISPHOSPHATE KINASE (IP5K) and INOSITOL HEXAKISPHOSPHATE KINASE 1 (IP6K1) pair modulates functions of Cullin RING Ubiquitin E3 ligases (CRLs) that execute targeted degradation of substrates. Coordinated activities of these kinases protect CRLs on a COP9 signalosome (CSN) platform and stimulates deneddylation-dependent disassembly to maintain continuity of its functions. In plants, CRL regulations on CSN by inositol phosphate (InsP) kinases are not known. Here, we show interactions of Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE 1 (IPK1) and INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), counterparts of the above InsP-kinase pair, with CSN subunits and its positive influences on the dynamics of cullin deneddylation. We identify neddylation enhancements on CRLs as an early response to phosphate-starvation and its orchestration by perturbed IPK1/ITPK...

The propensity for polyphosphorylation makes myo-inositol derivatives, the inositol polyphosphates (InsPs), especially phytic acid or inositol hexakisphosphate (InsP6) the major form of phosphate storage in plants. Acts of pyrophosphorylation on InsP6 generates InsP7 or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. Also implicated as co-factors for several phytohormone signaling networks, InsP7/InsP8 modulate key developmental processes. With recent identification as the common moeity for transducing both jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 is the classic example of a metabolite that may moonlight crosstalks to different cellular pathways during diverse stress adaptations. We show here that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2), but not other InsP-kinases, su...

Rapid adaptation of plants to developmental or physiological cues is facilitated by specific receptors that transduce the signals mostly via post-translational modification (PTM) cascades of downstream partners. Reversible covalent attachment of SMALL UBIQUITIN-LIKE MODIFIER (SUMO), a process termed as SUMOylation, influence growth, development and adaptation of plants to various stresses. Strong regulatory mechanisms maintain the steady-state SUMOylome and mutants with SUMOylation disturbances display mis-primed immunity often with growth consequences. Identity of the SUMO-substrates undergoing SUMOylation changes during defences however remain largely unknown. Here we exploit either the auto-immune property of an Arabidopsis mutant or defense responses induced in wild-type plants against Pseudomonas syringae pv tomato (PstDC3000) to enrich and identify SUMO1-substrates. Our results demonstrate massive enhancement of SUMO1-conjugates due to increased SUMOylation efficiencies during...

Reversible covalent attachment of SMALL UBIQUITIN-LIKE MODIFIERS (SUMOs) on target proteins regulate diverse cellular process across all eukaryotes. In Arabidopsis thaliana, most mutants with perturbed global SUMOylome display severe impairments in growth and adaptations to physiological stresses. Since SUMOs self-regulate activities of SUMOylation-associated proteins, existence of multiple isoforms introduces possibilities of their functional intersections which remain unexplored especially in plant systems. Using well-established defense responses elicited against virulent and avirulent Pseudomonas syringae pv. tomato strains, we investigated crosstalks in individual and combinatorial Arabidopsis sum mutants. Here we report that while SUM1 and SUM2 additively, but not equivalently suppress basal and TNL-specific immunity via down-regulation of salicylic acid (SA)-dependent responses, SUM3 promotes these defenses genetically downstream of SA. Remarkably, the expression of SUM3 is t...

Rice blast, caused by Magnaporthe oryzae (Couch & Kohn, 2002) is one of the most serious diseases of rice in all rice growing regions of the world. The disease can be effectively managed by the deployment of resistance (R) genes. Use of resistant rice varieties would be the most effective way to control this disease; therefore, mining the resistant genes might be important foundational work in the breeding program. India is the reservoir of rice biodiversity with huge number of traditional landraces. In the present study three well characterized genes, viz.,Pi1, Pi2 (Piz- 5) and Pi5 that are known to confer broad spectrum resistance to blast were mined from landrace collections of Karnataka state, India, by using PCR (polymerase chain reaction) based molecular markers. The results indicated the presence of Pi-1 gene in 39 landraces, accounting for 45.24% of 84 landraces analysed. Pi-z5and Pi-5 gene were present in 60 and 59 landraces accounting the distribution frequency of 71.43% a...

ABSTRACT Rice blast caused by Magnaporthe oryzae is the most devastating disease. Host-plant resistance is one of the important approaches to tackle this disease. India being the center of origin of rice, shelters numerous landraces adopted to overcome biotic and abiotic stresses. Three well characterized blast resistance genes, Pi1, Pi2 (Piz-5) and Pi5 were mined from rice landrace collection from Karnataka state, India by using gene linked and gene based molecular markers. Landraces were field screened for blast resistance. Pit gene was tracked in 39 landraces accounting 46.98% gene frequency. Sixty landraces contained Pi2 and Pi5 gene with 72.28% gene frequency. Twenty landrace harboured all the three genes.

In its lifetime a plant is exposed to pathogens of diverse types. Although methods of surveillance are broadly pathogen-individualized, immune signaling ultimately connect to common core networks maintained by key protein hubs. Defense elicitations modulate these hubs to re-allocate energy from central metabolic pathway into processes that execute immunity. Because unregulated defenses severely decrease growth and productivity of the host, signaling regulators within the networks function to achieve cellular equilibrium once the threat is minimized. Protein modifications by post-translational processes regulate the molecular switches and crosstalks between interconnected pathways spatially and temporally. Covalent modification of host targets connected to hubs are strategies used by most virulent effectors and result in re-routing signals to suppress host defenses. Resistance is a result of activation of specialized classes of receptors that short-circuit effector activities by co-localizing via post-translational modifications (PTMs) with effector targets. Despite advancement in proteome methodologies, our understanding of how PTMs regulate plant defenses remains elusive. This review presents protein-modifications as forefront regulators of plant innate immunity. © 2015 IUBMB Life, 67(7):524-532, 2015.