The role of phosphatidylinositol-specific phospholipase-C in plant defense signaling

Plant innate immunity requires immune receptors that sense the presence of microbes and activate defense reactions. Phosphatidylinositol-phospholipase C (PI-PLC) activity was previously shown to be important for several types of plant defenses although its signaling mechanism is not fully understood. It is also not clear why plants possess several PI-PLC isoforms and how triggering immune receptors activates them. PI-PLC activation induces a transient release of free cytosolic Ca2+ and the turnover of specific low abundant signaling phospholipids in the plasma membrane. Both events are important signals in animals and plants. Here, the first genetic evidence linking PI-PLC signaling to plant defense against pathogens is provided. The structure of the tomato PI-PLC family was determined, the corresponding genes were cloned and the function of individual isoforms during defense was studied. We found that PLC4 and PLC6 encode active enzymes and that they have distinct roles in defense. Optimum activity requirements and substrate preferences were determined for three PI-PLC enzymes and their enzyme activity was found to be important for immune receptor-activated responses. The available information was used to draw a model explaining the role of PI-PLC signaling in immune receptor-mediated defense and resistance in plants. </p

The tomato phosphatidylinositol-phospholipase C2 (SlPLC2) is required for defense gene induction by the fungal elicitor xylanase

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated upon pathogen attack. We have previously shown that the fungal elicitor xylanase rapidly induces nitric oxide (NO), which is required for PI-PLCs activity and downstream defense responses in tomato cell suspensions. Here, we show that all six SlPLC genes are expressed in tomato cell suspensions. Treatment of the cells with xylanase induces an early increase in SlPLC5 transcript levels, followed by a raise of the amount of SlPLC2 transcripts. The production of NO is required to augment SlPLC5 transcript levels in xylanase-treated tomato cells. Xylanase also induces SlPLC2 and SlPLC5 transcript levels in planta. We knocked-down the expression of SlPLC2 and SlPLC5 by virus-induced gene silencing. We found that SlPLC2 is required for xylanase-induced expression of the defense-related genes PR1 and HSR203J

Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity

Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCzeta. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca2+ ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors

An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1D481V, which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protei

An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1D481V, which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protei

Cytogenetic and molecular responses of ammonium sulphate application for tolerance to extreme temperatures in Vicia faba L.

Effects of ammonium sulphate [(NH4)(2)SO4] on mitosis, cell cycle and chromosomes in Vicia faba L. seeds exposed to extreme temperatures were investigated using flowcytometric and cytogenetic analysis. Seeds germinated at high and low temperatures showed a significant decrease in mitotic index as compared to those of optimum temperature conditions. Application of 50 and 1000 mu M (NH4)(2)SO4 were successful in alleviating the negative effects of low and high temperature on mitotic activity, respectively. 50 mu M (NH4)(2)SO4 showed the most positive effect on cell cycle at the extreme temperatures. This concentration increased the cell division removing or decreasing the negative effects of temperature stress. Namely, the highest G2/M and S phase percentages under stress conditions were obtained with application of 50 mu M (NH4)(2)SO4. Chromosomal aberrations were not observed in cells of seeds germinated in distilled water and also at any temperatures. However, the frequency of chromosomal aberrations increased significantly by increasing (NH4)(2)SO4 concentration. The highest aberration frequency in all temperature degree tested was found at 1000 mu M (NH4)(2)SO4 concentration.Department of Scientific Research Project Management of Suleyman Demirel University (SDUBAP)Suleyman Demirel University [1636-YL-08]The authors thank the Department of Scientific Research Project Management of Suleyman Demirel University (SDUBAP) for the financial support of the project SDUBAP (1636-YL-08). Thanks also to Dr. Gulderen Yanikkaya DEMIREL and Mehtap OZDEMIR (Istanbul Centro Laboratory Flowcytometry Department, Istanbul, Turkey) for its help in flow cytometric study