Necrotrophic Effectors SnTox from the Stagonospora nodorum (Berk.) Manipulate the Redox Metabolism of the Host Plant to Hijack Its Early Defense Response

Reactive oxygen species (ROS) play a central role in plant immune responses. The most important virulence factors of Stagonospora nodorum 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 in the early stages of infection has not been studied. In this study, our aim was to research the effect of S. nodorum effectors SnToxA, SnTox1, and SnTox3 on development of disease symptoms, the generation of hydrogen peroxide, and the enzyme activity of the redox metabolism in the early stage of infection in various wheat genotypes infected with isolates of S. nodorum (Sn4VD, SnB, and Sn9MN) carrying a set of various NE genes. Our results indicate that all three NEs of SnToxA, SnTox1, and SnTox3 play an important role in the inhibition of ROS in the initial stage of infection. Tsn1–SnToxA and Snn3–SnTox3 inhibited ROS production in wheat by affecting NADPH oxidases, peroxidases, superoxide dismutase, and catalase. Snn1–SnTox1 inhibited the production of ROS in wheat by mainly affecting peroxidase. NEs suppress ROS production only in the presence of the susceptibility genes Tsn1, Snn1, Snn3

Endophytic Strain Bacillus subtilis 26D Increases Levels of Phytohormones and Repairs Growth of Potato Plants after Colorado Potato Beetle Damage

Plant damage caused by defoliating insects has a long-term negative effect on plant growth and productivity. Consequently, the restoration of plant growth after exposure to pathogens or pests is the main indicator of the effectiveness of the implemented defense reactions. A short-term Leptinotarsa decemlineata Say attack on potato tube-grown plantlets (Solanum tuberosum L.) led to a reduction of both the length and mass of the shoots in 9 days. The decrease of the content of phytohormones—indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin and zeatin–riboside—in shoots of damaged potato plants was found. Endophytic strain Bacillus subtilis 26D (Cohn) is capable of secreting up to 83.6 ng/mL IAA and up to 150 ng/mL cytokinins into the culture medium. Inoculation of potato plants with cells of the B. subtilis 26D increases zeatin–riboside content in shoots and the mass of roots of undamaged plants, but does not influence content of IAA and ABA and growth of shoots. The presence of B. subtilis 26D in plant tissues promoted a rapid recovery of the growth rates of shoots, as well as the wet and dry mass of roots of plants after the pest attack, which we associate with the maintenance of a high level of IAA, ABA and cytokinins in their tissues

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

Regulation of Oncogene Expression in T-DNA-Transformed Host Plant Cells

Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance cytokinin and auxin levels for further cell proliferation

Novel Sources of Resistance to <i>Stagonospora nodorum</i> and Role of Effector-Susceptibility Gene Interactions in Wheat of Russian Breeding

Virulence factors of the pathogen Stagonospora nodorum Berk. are numerous necrotrophic effectors (NEs) (SnTox), which interact with the products of host susceptibility genes (Snn), causing the development of the disease. In this study, 55 accessions of bread spring and winter wheat were screened for sensitivity to NEs SnToxA, SnTox1, and SnTox3 using different isolates of S. nodorum. In the studied panel of wheat, 47 accessions were modern commercial cultivars grown in Russia and 8 cultivars were historic wheat accessions from the N. I. Vavilov Institute of Plant Genetic Resources in Russia. In general, our wheat panel differed from other wheat collections with available data in that it was less sensitive to SnToxA and SnTox3, and more sensitive to SnTox1. Six sources of strong SNB resistance were identified in our wheat panel. In addition, during the study, wheat cultivars were identified as appropriate objects in which to study the different effects of SnTox-Snn interactions, which is important for marker-assisted selection for SNB resistance. The current study has shown, for the first time, that the expression level of Snn1 and Tsn1 susceptibility genes and the disease severity of the different wheat cultivars are interconnected. Future work should focus on the deep characterization of SnTox-Snn interactions at the molecular level

The Role of Cytokinins and Abscisic Acid in the Growth, Development and Virulence of the Pathogenic Fungus <i>Stagonospora nodorum</i> (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

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

Ethylene-Cytokinin Interaction Determines Early Defense Response of Wheat against <i>Stagonospora nodorum</i> 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

Fast Degradation of Tetracycline and Ciprofloxacin in Municipal Water under Hydrodynamic Cavitation/Plasma with CeO2 Nanocatalyst

Although water contamination with drug residues is a threat to public health, there are currently barely any effective methods of purifying water from pharmaceutical substances. In this study, continuous-flow sonoplasma treatment was used for the complete degradation of tetracycline and ciprofloxacin in polluted municipal water. The addition of CeO2 nanoparticles as catalysts significantly increased the degradation rate of the antibiotics, and a degradation degree of 70% was achieved. The presence of reactive oxygen species in the CeO2-nanoparticle-containing sonoplasma-treated system was experimentally proven for the first time using the chemiluminescence technique

Additive Effect of the Composition of Endophytic Bacteria <i>Bacillus subtilis</i> on Systemic Resistance of Wheat against Greenbug Aphid <i>Schizaphis graminum</i> Due to Lipopeptides

The use of biocontrol agents based on endophytic bacteria against phloem-feeding insects is limited by a lack of knowledge and understanding of the mechanism of action of the endophyte community that makes up the plant microbiome. In this work, the mechanisms of the additive action of endophytic strains B. subtilis 26D and B. subtilis 11VM on the resistance of bread spring wheat against greenbug aphid Schizaphis graminum, was studied. It was shown that B. subtilis 26D secreted lipopeptide surfactin and phytohormones cytokinins, and B. subtilis 11VM produced iturin and auxins into the cultivation medium. Both strains and their lipopeptide-rich fractions showed direct aphicidal activity against greenbug aphid. For the first time, it was shown that B. subtilis 26D and B. subtilis 11VM in the same manner, as well as their lipopeptide-rich fractions, activated the expression of salicylate- and ethylene-dependent PR genes, and influenced plant redox metabolism, which led to an increase in plant endurance against aphids. The composition of endophytic strains B. subtilis 26D + B. subtilis 11VM had an additive effect on plant resistance to aphids due to an increase in the number of endophytic bacterial cells, and, as well as due to the synergistic effect of their mixture of lipopeptides − surfactin + iturin, both on the aphid mortality and on the expression of PR1 and PR3 genes. All these factors can be the reason for the observed increase in the growth of plants affected by aphids under the influence of B. subtilis 26D and B. subtilis 11VM, individually and in composition. The study demonstrates the possibility of creating in the future an artificial composition to enhance plant microbiome with endophytic bacteria, which combines growth-promoting and plant immunity stimulating properties against phloem-feeding insects. This direction is one of the most promising approaches to green pesticide discovery in the future