The Role of Cytokinins and Abscisic Acid in the Growth, Development and Virulence of the Pathogenic Fungus Stagonospora nodorum (Berk.).

Cytokinins (CKs) and abscisic acid (ABA) play an important role in the life of both plants and pathogenic fungi. However, the role of CKs and ABA in the regulation of fungal growth, development and virulence has not been sufficiently studied. We compared the ability of two virulent isolates (SnB and Sn9MN-3A) and one avirulent isolate (Sn4VD) of the pathogenic fungus Stagonospora nodorum Berk. to synthesize three groups of hormones (CKs, ABA and auxins) and studied the effect of exogenous ABA and zeatin on the growth, sporulation and gene expression of necrotrophic effectors (NEs) and transcription factors (TFs) in them. Various isolates of S. nodorum synthesized different amounts of CKs, ABA and indoleacetic acid. Using exogenous ABA and zeatin, we proved that the effect of these hormones on the growth and sporulation of S. nodorum isolates can be opposite, depends on both the genotype of the isolate and on the concentration of the hormone and is carried out through the regulation of carbohydrate metabolism. ABA and zeatin regulated the expression of fungal TF and NE genes, but correlation analysis of these parameters showed that this effect depended on the genotype of the isolate. This study will contribute to our understanding of the role of the hormones ABA and CKs in the biology of the fungal pathogen S. nodorum.

Virulence Factors of the Fungal Pathogen Stagonospora nodorum Manipulate Hormonal Signaling Pathways in Triticum aestivum L. by Regulating Host Plant MicroRNA Expressions.

BACKGROUND: Currently, the role of microRNAs in plant immune responses is being actively studied. Thus, our aim was to research the effect of Stagonospora nodorum (Berk.) NEs SnToxA and SnTox3 on the expression of miRNAs involved in the wheat-S. nodorum interaction and to determine the role of phytohormones in this process. METHODS: The expressions of nine conserved microRNAs were studied by quantitative real-time polymerase chain reaction in three different wheat genotypes of bread spring wheat (Triticum aestivum L.) infected with S. nodorum. Phytohormone treatments (trans-zeatin, 2-chloroethylphosphonic acid (etefone is the chemical precursor of ethylene), and salicylic acid) were applied. The results were compared with disease symptoms, the redox status of plants, and the expression of fungal necrotrophic effector (NE) genes of SnToxA and SnTox3 and genes of SnPf2, SnStuA, alongside SnCon7 transcription factors (TFs). RESULTS: Salicylic acid (SA) and cytokinins (CK) are involved in the development of defense reactions in wheat plants against S. nodorum, by regulating the expression of fungal NEs and TFs genes, inducing an oxidative burst in all three wheat genotypes. Moreover, ethylene enhanced the virulence of the pathogen by increasing the expression of fungal NE and TF genes, thereby resulting in a decrease in the generation of reactive oxygen species in all three cultivars. The nine miRNAs played a role in the development of wheat resistance against S. nodorum. NE SnTox3 mainly suppressed the expression of three miRNAs: miR159, miR393, and miR408, while NE SnToxA suppressed miR166 expression. Conversely, treatment with CK and SA increased the expression of miR159 and miR408; treatment with CK increased the expression of miR393 and miR166. Ethylene inhibited the expression of miR159, miR408, miR393, and miR166. Suppression of miP159 expression by NE SnTox3 was most likely associated with the activation of the ethylene signaling pathway. NEs SnToxA and SnTox3 suppressed the expression of miR408, whose role most likely consisted of inhibiting the catalase activity, via SA and CK regulation. In addition, NE SnToxA hijacked the SA signaling pathway and manipulated it for fungal growth and development. Fungal TFs SnPf2 and SnStuA could be involved in the regulation of these processes indirectly through the regulation of the expression of NE genes. CONCLUSIONS: The results of this work show, for the first time, the role of microRNAs in the development of wheat resistance against S. nodorum and the effect of S. nodorum NEs SnToxA and SnTox3 on the activity of plant microRNAs.

The Effects of Rhizosphere Inoculation with Pseudomonas mandelii on Formation of Apoplast Barriers, HvPIP2 Aquaporins and Hydraulic Conductance of Barley.

Pseudomonas mandelii strain IB-Ki14 has recently been shown to strengthen the apoplastic barriers of salt-stressed plants, which prevents the entry of toxic sodium. It was of interest to find out whether the same effect manifests itself in the absence of salinity and how this affects the hydraulic conductivity of barley plants. Berberine staining confirmed that the bacterial treatment enhanced the deposition of lignin and suberin and formation of Casparian bands in the roots of barley plants. The calculation of hydraulic conductance by relating transpiration to leaf water potential showed that it did not decrease in bacteria-treated plants. We hypothesized that reduced apoplastic conductivity could be compensated by the higher conductivity of the water pathway across the membranes. This assumption was confirmed by the results of the immunolocalization of HvPIP2;5 aquaporins with specific antibodies, showing their increased abundance around the areas of the endodermis and exodermis of bacteria-treated plants. The immunolocalization with antibodies against auxins and abscisic acid revealed elevated levels of these hormones in the roots of plants treated with bacteria. This root accumulation of hormones is likely to be associated with the ability of Pseudomonas mandelii IB-Ki14 to synthesize these hormones. The involvement of abscisic acid in the control of aquaporin abundance and auxins-in the regulation of and formation of apoplast barriers-is discussed.

Effects of Salinity and Abscisic Acid on Lipid Transfer Protein Accumulation, Suberin Deposition and Hydraulic Conductance in Pea Roots.

Lipid transfer proteins (LTPs) participate in many important physiological processes in plants, including adaptation to stressors, e.g., salinity. Here we address the mechanism of this protective action of LTPs by studying the interaction between LTPs and abscisic acid (ABA, a "stress" hormone) and their mutual participation in suberin deposition in root endodermis of salt-stressed pea plants. Using immunohistochemistry we show for the first time NaCl induced accumulation of LTPs and ABA in the cell walls of phloem paralleled by suberin deposition in the endoderm region of pea roots. Unlike LTPs which were found localized around phloem cells, ABA was also present within phloem cells. In addition, ABA treatment resulted in both LTP and ABA accumulation in phloem cells and promoted root suberization. These results suggested the importance of NaCl-induced accumulation of ABA in increasing the abundance of LTPs and of suberin. Using molecular modeling and fluorescence spectroscopy we confirmed the ability of different plant LTPs, including pea Ps-LTP1, to bind ABA. We therefore hypothesize an involvement of plant LTPs in ABA transport (unloading from phloem) as part of the salinity adaptation mechanism.

Growth-Promoting Effect of Rhizobacterium (Bacillus subtilis IB22) in Salt-Stressed Barley Depends on Abscisic Acid Accumulation in the Roots.

An ABA-deficient barley mutant (Az34) and its parental cultivar (Steptoe) were compared. Plants of salt-stressed Az34 (100 mmol m-3 NaCl for 10 days) grown in sand were 40% smaller than those of "Steptoe", exhibited a lower leaf relative water content and lower ABA concentrations. Rhizosphere inoculation with IB22 increased plant growth of both genotypes. IB22 inoculation raised ABA in roots of salt-stressed plants by supplying ABA exogenously and by up-regulating ABA synthesis gene HvNCED2 and down-regulating ABA catabolic gene HvCYP707A1. Inoculation partially compensated for the inherent ABA deficiency of the mutant. Transcript abundance of HvNCED2 and related HvNCED1 in the absence of inoculation was 10 times higher in roots than in shoots of both mutant and parent, indicating that ABA was mainly synthesized in roots. Under salt stress, accumulation of ABA in the roots of bacteria-treated plants was accompanied by a decline in shoot ABA suggesting bacterial inhibition of ABA transport from roots to shoots. ABA accumulation in the roots of bacteria-treated Az34 was accompanied by increased leaf hydration, the probable outcome of increased root hydraulic conductance. Thereby, we tested the hypothesis that the ability of rhizobacterium (Bacillus subtilis IB22) to modify responses of plants to salt stress depends on abscisic acid (ABA) accumulating in roots.

Involvement of Reactive Oxygen Species in ABA-Induced Increase in Hydraulic Conductivity and Aquaporin Abundance.

The role of reactive oxygen species (ROS) in ABA-induced increase in hydraulic conductivity was hypothesized to be dependent on an increase in aquaporin water channel (AQP) abundance. Single ABA application or its combination with ROS manipulators (ROS scavenger ascorbic acid and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI)) were studied on detached roots of barley plants. We measured the osmotically driven flow rate of xylem sap and calculated root hydraulic conductivity. In parallel, immunolocalization of ABA and HvPIP2;2 AQPs was performed with corresponding specific antibodies. ABA treatment increased the flow rate of xylem, root hydraulic conductivity and immunostaining for ABA and HvPIP2;2, while the addition of antioxidants prevented the effects of this hormone. The obtained results confirmed the involvement of ROS in ABA effect on hydraulic conductivity, in particular, the importance of H2O2 production by ABA-treated plants for the effect of this hormone on AQP abundance.

Reactive Oxygen Species in Host Plant Are Required for an Early Defense Response against Attack of Stagonospora nodorum Berk. Necrotrophic Effectors SnTox.

Reactive oxygen species (ROS) play a central role in plant immune responses. The most important virulence factors of the Stagonospora nodorum Berk. are multiple fungal necrotrophic effectors (NEs) (SnTox) that affect the redox-status and cause necrosis and/or chlorosis in wheat lines possessing dominant susceptibility genes (Snn). However, the effect of NEs on ROS generation at the early stages of infection has not been studied. We studied the early stage of infection of various wheat genotypes with S nodorum isolates -Sn4VD, SnB, and Sn9MN, carrying a different set of NE genes. Our results indicate that all three NEs of SnToxA, SnTox1, SnTox3 significantly contributed to cause disease, and the virulence of the isolates depended on their differential expression in plants (Triticum aestivum L.). The Tsn1-SnToxA, Snn1-SnTox1and Snn3-SnTox3 interactions played an important role in inhibition ROS production at the initial stage of infection. The Snn3-SnTox3 inhibited ROS production in wheat by affecting NADPH-oxidases, peroxidases, superoxide dismutase and catalase. The Tsn1-SnToxA inhibited ROS production in wheat by affecting peroxidases and catalase. The Snn1-SnTox1 inhibited the production of ROS in wheat by mainly affecting a peroxidase. Collectively, these results show that the inverse gene-for gene interactions between effector of pathogen and product of host sensitivity gene suppress the host's own PAMP-triggered immunity pathway, resulting in NE-triggered susceptibility (NETS). These results are fundamentally changing our understanding of the development of this economical important wheat disease.

Ethylene-Cytokinin Interaction Determines Early Defense Response of Wheat against Stagonospora nodorum Berk.

Ethylene, salicylic acid (SA), and jasmonic acid are the key phytohormones involved in plant immunity, and other plant hormones have been demonstrated to interact with them. The classic phytohormone cytokinins are important participants of plant defense signaling. Crosstalk between ethylene and cytokinins has not been sufficiently studied as an aspect of plant immunity and is addressed in the present research. We compared expression of the genes responsible for hormonal metabolism and signaling in wheat cultivars differing in resistance to Stagonospora nodorum in response to their infection with fungal isolates, whose virulence depends on the presence of the necrotrophic effector SnTox3. Furthermore, we studied the action of the exogenous cytokinins, ethephon (2-chloroethylphosphonic acid, ethylene-releasing agent) and 1-methylcyclopropene (1-MCP, inhibitor of ethylene action) on infected plants. Wheat susceptibility was shown to develop due to suppression of reactive oxygen species production and decreased content of active cytokinins brought about by SnTox3-mediated activation of the ethylene signaling pathway. SnTox3 decreased cytokinin content most quickly by its activated glucosylation in an ethylene-dependent manner and, furthermore, by oxidative degradation and inhibition of biosynthesis in ethylene-dependent and ethylene-independent manners. Exogenous zeatin application enhanced wheat resistance against S. nodorum through inhibition of the ethylene signaling pathway and upregulation of SA-dependent genes. Thus, ethylene inhibited triggering of SA-dependent resistance mechanism, at least in part, by suppression of the cytokinin signaling pathway.

Integrating edge detection and fuzzy connectedness for automated segmentation of anatomical branching structures.

Image segmentation algorithms are critical components of medical image analysis systems. This paper presents a novel and fully automated methodology for segmenting anatomical branching structures in medical images. It is a hybrid approach which integrates the Canny edge detection to obtain a preliminary boundary of the structure and the fuzzy connectedness algorithm to handle efficiently the discontinuities of the returned edge map. To ensure efficient localisation of weak branches, the fuzzy connectedness framework is applied in a sliding window mode and using a voting scheme the optimal connection point is estimated. Finally, the image regions are labelled as tissue or background using a locally adaptive thresholding technique. The proposed methodology is applied and evaluated in segmenting ductal trees visualised in clinical X-ray galactograms and vasculature visualised in angiograms. The experimental results demonstrate the effectiveness of the proposed approach achieving high scores of detection rate and accuracy among state-of-the-art segmentation techniques.