Redox and Hormonal Changes in the Transcriptome of Grape (Vitis vinifera) Berries during Natural Noble Rot Development.

Noble rot is a favorable form of the interaction between grape (Vitis spp.) berries and the phytopathogenic fungus Botrytis cinerea. The transcriptome pattern of grapevine cells subject to natural noble rot development in the historic Hungarian Tokaj wine region has not been previously published. Furmint, a traditional white Tokaj variety suited to develop great quality noble rot was used in the experiments. Exploring a subset of the Furmint transcriptome redox and hormonal changes distinguishing between noble rot and bunch rot was revealed. Noble rot is defined by an early spike in abscisic acid (ABA) accumulation and a pronounced remodeling of ABA-related gene expression. Transcription of glutathione S-transferase isoforms is uniquely upregulated, whereas gene expression of some sectors of the antioxidative apparatus (e.g., catalases, carotenoid biosynthesis) is downregulated. These mRNA responses are lacking in berries exposed to bunch rot. Our results help to explain molecular details behind the fine and dynamic balance between noble rot and bunch rot development.

Road-killed mammals provide insight into tick-borne bacterial pathogen communities within urban habitats.

Small- and medium-sized mammals play an important role in the life cycle of tick-borne pathogens in urban habitats. Our aim was to apply the general protocol, DAMA (documentation-assessment-monitoring-action), which is an integrated proposal to build a proactive capacity to understand, anticipate, and respond to the outcomes of accelerating environmental change. Here we tested whether road-killed carcasses in urban areas are useful sources of tissue and parasite samples to investigate these species' contribution to the epidemiology of vector-borne diseases. We collected 29 road-killed and 6 carcasses with different causes of mortality (23 northern white-breasted hedgehogs and 12 from seven other mammal species) mainly from Budapest, Hungary. We used quantitative and conventional PCRs to determine pathogens in 90 collected tissues (52 from hedgehogs; 38 from other species) and 417 ticks that were only found on hedgehogs. Tissue samples revealed a wide range of bacteria including human zoonotic pathogens identified as Anaplasma phagocytophilum ecotype I, Borrelia afzelii, B. spielmanii, Borrelia miyamotoi, Rickettsia helvetica, and Bartonella species. Among the 23 collected hedgehog carcasses, 17 (74%) were infected with A. phagocytophilum, 6 (26%) with Borrelia burgdorferi s.l., 12 (52%) with R. helvetica, and 15 (65%) with Rickettsia sp. Furthermore, we report the first detection of Rickettsia sp. infection in European moles and lesser weasel and R. helvetica in stone marten. Through sequencing B. afzelii, R. helvetica, R. monacensis and A. phagocytophilum ecotype I were identified in the ticks removed from the carcasses. We showed that road-killed urban mammal species are exposed to multiple tick-borne pathogens but further studies have to clarify whether they, in fact, also have a role in their maintenance and spread. Our study also demonstrates that roadkill can be used in the risk assessment of potential human infection and in the implementation of the DAMA protocol.

Contribution of cell wall peroxidase- and NADPH oxidase-derived reactive oxygen species to Alternaria brassicicola-induced oxidative burst in Arabidopsis.

Cell wall peroxidases and plasma membrane-localized NADPH oxidases are considered to be the main sources of the apoplastic oxidative burst in plants attacked by microbial pathogens. In spite of this established doctrine, approaches attempting a comparative, side-by-side analysis of the functions of extracellular reactive oxygen species (ROS) generated by the two enzymatic sources are scarce. Previously, we have reported the role of Arabidopsis NADPH oxidase RBOHD (respiratory burst oxidase homologue D) in plants challenged with the necrotrophic fungus Alternaria brassicicola. Here, we present results on the activity of apoplastic class III peroxidases PRX33 (At3g49110) and PRX34 (At3g49120) investigated in the same Arabidopsis-Alternaria pathosystem. ROS generated by Arabidopsis peroxidases PRX33 and PRX34 increase the necrotic symptoms and colonization success of A. brassicicola. In addition, the knockdown of PRX33 and PRX34 transcript levels leads to a reduced number of host cells showing an extracellular burst of ROS after inoculation with A. brassicicola. Our results also reveal an age-dependent transcript distribution of ROS-producing peroxidase and NADPH oxidase enzymes, and some potential new components of the RBOHD, PRX33 and PRX34 signalling networks.

A simplified and efficient Agrobacterium tumefaciens electroporation method.

Agrobacterium tumefaciens is a widely used microbial tool in plant molecular biology to transfer DNA into plant cells and produce, e.g., stable or transient transformants or induce gene silencing. In our study, we present a simplified version of electrocompetent cell preparation that is not only time and cost efficient, but it requires minimal handling of bacterial cells. Liquid cultures are normally used to prepare competent Agrobacterium cells. To overcome the difficulties of working with liquid cultures, we propose suspending bacterial cells directly from overnight agar plate cultures. In addition, we optimized several parameters to simplify the procedure and maximize the number of transformants (e.g., Agrobacterium strains, number of washing steps, amount of required plasmid DNA, electroporation parameters, type of incubation media, or incubation time). This optimized, simple, and fast protocol has proved to be efficient enough to obtain transformed colonies with low amounts (as little as 1 ng) of plasmid DNA. In addition, it also enabled us to introduce ligated plasmids directly into Agrobacterium omitting the E. coli transformation step and accelerating the cloning procedure further.

Description of the Nicotiana benthamiana-Cercospora nicotianae Pathosystem.

Nicotiana benthamiana is a valuable model organism in plant biology research. This report describes its extended applicability in the field of molecular plant pathology by introducing a nonbiotrophic fungal pathogen Cercospora nicotianae that can be conveniently used under laboratory conditions, consistently induces a necrotic leaf spot disease on Nicotiana benthamiana, and is specialized on solanaceous plants. Our inoculation studies showed that C. nicotianae more effectively colonizes N. benthamiana than its conventional host, N. tabacum. The functions of two critical regulators of host immunity, coronatine-insensitive 1 (COI1) and ethylene-insensitive 2 (EIN2), were studied in N. benthamiana using Tobacco rattle virus-based virus-induced gene silencing (VIGS). Perturbation of jasmonic acid or ethylene signaling by VIGS of either COI1 or EIN2, respectively, resulted in markedly increased Cercospora leaf spot symptoms on N. benthamiana plants. These results suggest that the N. benthamiana-C. nicotianae host-pathogen interaction is a prospective but hitherto unutilized pathosystem for studying gene functions in diseased plants.

Overlapping Yet Response-Specific Transcriptome Alterations Characterize the Nature of Tobacco-Pseudomonas syringae Interactions.

In this study transcriptomic alterations of bacterially induced pattern triggered immunity (PTI) were compared with other types of tobacco-Pseudomonas interactions. In addition, using pharmacological agents we blocked some signal transduction pathways (Ca(2+) influx, kinases, phospholipases, proteasomic protein degradation) to find out how they contribute to gene expression during PTI. PTI is the first defense response of plant cells to microbes, elicited by their widely conserved molecular patterns. Tobacco is an important model of Solanaceae to study resistance responses, including defense mechanisms against bacteria. In spite of these facts the transcription regulation of tobacco genes during different types of plant bacterial interactions is not well-described. In this paper we compared the tobacco transcriptomic alterations in microarray experiments induced by (i) PTI inducer Pseudomonas syringae pv. syringae type III secretion mutant (hrcC) at earlier (6 h post inoculation) and later (48 hpi) stages of defense, (ii) wild type P. syringae (6 hpi) that causes effector triggered immunity (ETI) and cell death (HR), and (iii) disease-causing P. syringae pv. tabaci (6 hpi). Among the different treatments the highest overlap was between the PTI and ETI at 6 hpi, however, there were groups of genes with specifically altered activity for either type of defenses. Instead of quantitative effects of the virulent P. tabaci on PTI-related genes it influenced transcription qualitatively and blocked the expression changes of a special set of genes including ones involved in signal transduction and transcription regulation. P. tabaci specifically activated or repressed other groups of genes seemingly not related to either PTI or ETI. Kinase and phospholipase A inhibitors had highest impacts on the PTI response and effects of these signal inhibitors on transcription greatly overlapped. Remarkable interactions of phospholipase C-related pathways with the proteasomal system were also observable. Genes specifically affected by virulent P. tabaci belonged to various previously identified signaling routes, suggesting that compatible pathogens may modulate diverse signaling pathways of PTI to overcome plant defense.

Regulatory Proteolysis in Arabidopsis-Pathogen Interactions.

Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is simultaneously used both by plant cells, to recognize and inactivate invading pathogens, and by microbes, to overcome the immune system of the plant and successfully colonize host cells. In this review, we present available results on the group of proteases in the model plant Arabidopsis thaliana whose functions in microbial pathogenesis were confirmed. Pathogen-derived proteolytic factors are also discussed when they are involved in the cleavage of host metabolites. Considering the wealth of review papers available in the field of the ubiquitin-26S proteasome system results on the ubiquitin cascade are not presented. Arabidopsis and its pathogens are conferred with abundant sets of proteases. This review compiles a list of those that are apparently involved in an interaction between the plant and its pathogens, also presenting their molecular partners when available.