Biology subjects

Tsakiri, D.

Publications and source records attributed to Tsakiri, D..

3 recordsLinked to original sources

bioRxiv2022

The functional and structural characterization of Xanthomonas campestris pv. campestris core effector XopP revealed a new kinase activity

The exocyst complex subunit protein Exo70B1 plays a crucial role in a variety of cell mechanisms including immune responses against pathogens. The calcium dependent kinase 5 (CPK5) of Arapidopsis thaliana, phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestis effector XopP, compromises Exo70B1 and bypasses the hosts hypersensitive response (HR), in a way that is still unclear. Herein we designed an experimental approach based on biophysical, biochemical and molecular assays, based on structural and functional predictions, as well as, utilizing Aplhafold and DALI online servers respectively, in order to characterize the in vivo XccXopP function. The interaction between AtExo70B1 and XccXopP is very stable in high temperatures, while the AtExo70B1 appeared to be phosphorylated at XccXopP expressing transgenic Arabidopsis. XccXopP reveals similarities with known mammalian kinases, and phosphorylates AtExo70B1 at Ser107, Ser111, Ser248, Thr309 and Thr364. Furthermore, XccXopP protects AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that, XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its hosts protein target AtExo70B1, but also protects the latter from the innate AtCPK5 phosphorylation, to bypass the hosts immune responses.

microbiology↗

bioRxiv2022

Ralstonia solanacearum core effector RipE1 interacts and cleaves the Arabidopsis exocyst component Exo70B1

Ralstonia solanacearum depends on numerous virulence factors, also known as effectors, to promote disease in a wide range of economically important host plants. Although some of these effectors have been characterized, none have yet been shown to target the hosts secretion machinery. Here, we used an extended library of NLR plant immune receptor integrated domains (IDs), to identify new effector targets. The screen uncovered that the core effector RipE1, of the R. solanacearum species complex, among other targets, associates with Arabidopsis exocyst component Exo70B1. RipE1, in accordance with its predicted cysteine protease activity, cleaves Exo70B1 in vitro and also promotes Exo70B1 degradation in planta. RipE1 enzymatic activity additionally results in the activation of TN2-dependent ectopic cell death. TN2 is an atypical NLR that has been proposed to guard Exo70B1. Despite the fact that RipE1 has been previously reported to activate defense responses in model plant species, we present here a Nicotiana species, in which RipE1 expression does not activate cell death. In addition, we discovered that RipE1 is recognized by Ptr1, a Nicotiana benthamiana CC-NLR, via its cysteine protease activity. Overall, this study uncovers a new RipE1 host target and a new RipE1-activated NLR while providing evidence and novel tools to advance in-depth studies of RipE1 and homologous effectors. Author SummaryBacterial wilt disease caused by Ralstonia solanacearum, poses a serious global threat for a wide range of agriculturally important plant species. This Gram-negative bacterium utilizes a collection of Type III Secretion System (T3SS) effectors to manipulate host cell defense and physiology. In this study, we searched for new subcellular plant targets of the core R. solanacearum effector RipE1, a cysteine protease. We discovered that RipE1 has multiple potential eukaryotic targets and further elucidated its association with the host exocyst complex. Using Artificial Intelligence (AI)-based predictions and performing both in vitro and in planta assays, we found that RipE1 promotes the degradation of plant exocyst component Exo70B1 through its enzymatic activity. Apart from being the first report of a R. solanacearum effector targeting a component of the host secretion machinery, our findings also identify an NLR from a model plant species that is able to recognize RipE1 protease activity and provide evidence that can lead to the discovery of additional RipE1 targets inside the host cell.

microbiology↗

bioRxiv2021

Expression of putative effectors of different Xylella fastidiosa subspecies/strains reveals recognition and defense activation in various model plants

The re-emergence of Gram-negative bacterium Xylella fastidiosa in Europe in 2013 impelled the scientific community to discover novel strategies for crop protection. The wide host range of Xylella indicates the existence of yet not characterized pathogenic mechanisms to overcome plant defenses. The recent uprising accuracy of a variety of bioinformatics tools, with the ability to predict the function of putative microbial protein represent a useful approach for understanding which of these proteins are associated with pathogens virulence. In this study we collected a number of putative effectors from two X. fastidiosa strains: Temecula1 and CoDiRo and the subspecies (ssp.) Sandyi Ann-1. We designed an in-planta Agrobacterium based expression system that drives the expressed proteins to the cell apoplast, in order to investigate their ability to activate defense in various model plants. Furthermore, we organized the resulted proteins according to their sequential and structural similarities via the I-TASSER online tool. We identified that various X. fastidiosa proteins were able to differentially elicit cell death-like phenotypes in Nicotiana tabacum, N. sylvestris and N. benthamiana. These proteins are members of different enzymatic groups: a) hydrolases/hydrolases inhibitors, b) serine proteases and c) metal transferases. Collectively, we identified structurally similar proteins that were able to differentially elicit cell death-like phenotypes in different cultivars of the same species. Our findings provide the bases for further studies on the mechanisms that underlie host-defense activation by X. fastidiosa putative effectors, as well as, pathogens adaptation in susceptible hosts.

microbiology↗