BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF THE BACTERIAL PsaR1 SENSOR IN THE PSEUDOMONAS SYRINGAE PV. ACTINIDIAE-KIWIFRUIT INTERACTION

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is responsible for important economic losses in all major areas of kiwifruit production worldwide, including Italy. As for many other bacterial diseases, current plant defense strategies against Psa are mainly based on the use of copper-containing products, which raise eco-toxicological problems. Revision and restriction processes regarding the use of high amounts of copper in agriculture impose an urgent study of new solutions, efficient and eco-compatible, avoiding at the same time the occurrence of new resistance to active molecules. Innovative strategies are based for instance on the application of targeted treatments for \u201cweakening\u201d the pathogen, i.e. to reduce its virulence within its host. However, this requires improving our knowledge regarding molecular mechanisms controlling bacterial virulence induction. A key regulator of bacterial virulence is the so-called \u2018quorum-sensing\u2019 (QS), that links bacterial density to gene expression. This mechanism allows bacteria to communicate within the bacterial community and with their environment, via small diffusible molecules. The prototypical QS system of Gram-negative bacteria consists of a LuxI-type synthase that produces the signal molecules acyl homoserine lactones (AHLs) and a cognate LuxR-type receptor/regulator that senses signal specific threshold concentration. An interesting subgroup of LuxR receptors lacks a genetically linked LuxI and has been termed \u201csolos\u201d. These \u201csolos\u201d are assumed to sense AHLs from neighboring bacteria, bacterial molecules other than AHLs or still unknown plant-produced compounds in the case of phytopathogenic bacteria. Interestingly, Psa does not produce AHLs but possesses three LuxR solos, which likely contribute to Psa virulence. As a first candidate for a targeted inhibition strategy against Psa, we are currently investigating the biochemical properties of the sensor PsaR1. To that purpose, several tentative have been made to obtain the soluble recombinant sensor in a heterologous system. Once achieved, we demonstrated that it does not bind AHLs, thus excluding the possibility to sense AHLs from neighboring bacteria, and we are currently setting a chemical screening, based on thermal shift assay, to identify the class(es) of molecules able to bind to the sensor. On the other hand, we aim to identify the pathway(s) regulated by PsaR1 during Psa interaction with kiwifruit, during different phases of the infection. Thus, a microarray analysis is being performed to compare the transcriptomic profiles of wild-type and psaR1 knockout Psa strains at both exponential and stationary growth phase, in conditions mimicking the interaction with the host plant, i.e. minimal medium supplemented with kiwifruit extract

Detection of Peroxynitrite in Plants Exposed to Bacterial Infection

Peroxynitrite is a highly reactive derivative of nitric oxide (NO) which is gaining attention in the plant biology community because it may play a role in NO signaling during biotic stress. Peroxynitrite can react with many different biomolecules, but its ability to nitrate the tyrosine residues of proteins is particularly important because this may regulate defense signaling in response to pathogens. The analysis of peroxynitrite levels in the context of its proposed defense role requires an accurate and specific detection method. Here, we describe a photometric assay using the fluorescent dye Hong Kong Green 2 as a specific and quantitative probe for peroxynitrite in Arabidopsis thaliana plants challenged with an avirulent strain of Pseudomonas syringae pv. tomato. This protocol includes the preparation of plant samples, the assay procedure, the measurement of peroxynitrite-specific fluorescence, and data presentation

Detection and function of nitric oxide during the hypersensitive response in Arabidopsis thaliana: where there's a will there's a way

Nitric oxide (NO) was identified as a key player in plant defence responses approximately 20\u2009years ago and a large body of evidence has accumulated since then supporting its role as a signalling molecule. However, there are many discrepancies in current NO detection assays and the enzymatic pathways responsible for its synthesis have yet to be determined. This has provoked strong debates concerning the function of NO in plants, even questioning its existence in planta. Here we gather data obtained using the model pathosystem Arabidopsis/Pseudomonas, which confirms the production of NO during the hypersensitive response and supports is role as a trigger of hypersensitive cell death and a mediator of defence gene expression. Finally, we discuss potential sources of NO synthesis, focusing on the role of nitrite as major substrate for NO production during incompatible interactions

Nitric Oxide Signaling during the Hypersensitive Disease Resistance Response.

Nitric oxide (NO) signaling is known to play a key role in triggering the hypersensitive response (HR) of plants to avirulent pathogens. In this chapter, we have summarized what is currently known about the role of NO in this important biological context. We have discussed NO production and turnover leading to the accumulation of this reactive compound when plants are challenged by pathogens. Unfortunately, enzymatic system for its production and the molecular basis for its accumulation are still largely unknown. Furthermore, we have reviewed the ways by which NO transduces its activity to establish hypersensitive disease resistance response by discussing novel emerging findings about its functions. By considering characterized NO targets, we have shown that NO signaling mainly relies on its reactivity with diverse protein targets. This finally orchestrates the crosstalk of NO with other signaling pathways and modulates defence gene expression, allowing the execution of HR cell death and the triggering of the defence respons

Protein nitration during defence response in Arabidopsis thaliana

Nitric oxide and reactive oxygen species play a key role in the plant hypersensitive disease resistance response, and protein tyrosine nitration is emerging as an important mechanism of their co-operative interaction. Up to now, the proteins targeted by this post-translational modification in plants are still totally unknown. In this study, we analyzed for the first time proteins undergoing nitration during the hypersensitive response by analyzing via 1D- and 2D-western blot the protein extracts from Arabidopsis thaliana plants challenged with an avirulent bacterial pathogen (Pseudomonas syringae pv. Tomato). We show that the plant disease resistance response is correlated with a modulation of nitration of proteins involved in important cellular process, such as photosynthesis, glycolysis and nitrate assimilation. These findings shed new light on the signaling functions of nitric oxide and reactive oxygen species, paving the way on studies on the role of this post-translational modification in plants

Application of Chemical Genomics to Plant-Bacteria Communication: A High-Throughput System to Identify Novel Molecules Modulating the Induction of Bacterial Virulence Genes by Plant Signals

The life cycle of bacterial phytopathogens consists of a benign epiphytic phase, during which the bacteria grow in the soil or on the plant surface, and a virulent endophytic phase involving the penetration of host defenses and the colonization of plant tissues. Innovative strategies are urgently required to integrate copper treatments that control the epiphytic phase with complementary tools that control the virulent endophytic phase, thus reducing the quantity of chemicals applied to economically and ecologically acceptable levels. Such strategies include targeted treatments that weaken bacterial pathogens, particularly those inhibiting early infection steps rather than tackling established infections. This chapter describes a reporter gene-based chemical genomic high-throughput screen for the induction of bacterial virulence by plant molecules. Specifically, we describe a chemical genomic screening method to identify agonist and antagonist molecules for the induction of targeted bacterial virulence genes by plant extracts, focusing on the experimental controls required to avoid false positives and thus ensuring the results are reliable and reproducible

Identification, characterization, and expression analysis of calmodulin and calmodulin-like genes in grapevine (Vitis vinifera) reveal likely roles in stress responses

Calcium (Ca2+) is an ubiquitous key second messenger in plants, where it modulates many developmental and adaptive processes in response to various stimuli. Several proteins containing Ca2+ binding domain have been identified in plants, including calmodulin (CaM) and calmodulin-like (CML) proteins, which play critical roles in translating Ca2+ signals into proper cellular responses. In this work, a genome-wide analysis conducted in Vitis vinifera identified three CaM- and 62 CML-encoding genes. We assigned gene family nomenclature, analyzed gene structure, chromosomal location and gene duplication, as well as protein motif organization. The phylogenetic clustering revealed a total of eight subgroups, including one unique clade of VviCaMs distinct from VviCMLs. VviCaMs were found to contain four EF-hand motifs whereas VviCML proteins have one to five. Most of grapevine CML genes were intronless, while VviCaMs were intron rich. All the genes were well spread among the 19 grapevine chromosomes and displayed a high level of duplication. The expression profiling of VviCaM/VviCML genes revealed a broad expression pattern across all grape organs and tissues at various developmental stages, and a significant modulation in biotic stress-related responses. Our results highlight the complexity of CaM/CML protein family also in grapevine, supporting the versatile role of its different members in modulating cellular responses to various stimuli, in particular to biotic stresses. This work lays the foundation for further functional and structural studies on specific grapevine CaMs/CMLs in order to better understand the role of Ca2+-binding proteins in grapevine and to explore their potential for further biotechnological applications