Re‐targeting of a plant defense protease by a cyst nematode effector

Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector‐coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two‐hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain‐like cysteine protease (PLCP) ‘Responsive to Dehydration 21A’ (RD21A), which has been shown to function in the plant defense response. Activity‐based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re‐localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two‐hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector‐mediated re‐localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense‐inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.     

Root colonization and iron nutritional status of a Pseudomonas fluorescens in different plant species

Root colonization and induction of an iron stress regulated promoter for siderophore production by Pseudomonas fluorescens 2-79RLI was studied in vitro and in the rhizosphere of different plant species. P. fluorescens 2-79RLI was previously genetically modified with an iron regulated ice nucleation reporter, which allowed calibration of ice nucleation activity with siderophore production. Initial experiments examined ice nucleation activity and siderophore production under different growth conditions in vitro. These studies demonstrated that P. fluorescens 2-79RLI could utilize both Fe-citrate and Fe-phytosiderophore as iron sources, suggesting that production of these compounds by plants would increase iron availability for P. fluorescens 2-79RLI in the rhizosphere. Fe demand and Fe stress were further shown to be a function of nutrient availability and were reduced when carbon was limiting for growth. Subsequent experiments extended these observations to rhizosphere cells. Cells were sampled from the rhizosphere and the rhizoplane. Results of a soil microcosm experiment showed that Fe stress was reduced for P. fluorescens 2-79RLI in the barley rhizosphere as compared to the cells in the rhizosphere.of lupin. In lupin, relative Fe stress of P. fluorescens 2-79RLI was greater at the root tip than in the lateral root zone. In a second experiment comparing zucchini and bean, iron stress was greater for P. fluorescens 2-79RLI associated with zucchini than with bean. In a third experiment with rape plants under P deficient conditions, addition of soluble P was shown to increase Fe stress for P. fluorescens 2-79RLI located at the root tip, but not in the lateral root zone. This study showed that Fe stress of P. fluorescens 2-79RLI in the rhizosphere may be influenced by plant species, P source, root zone and localization of the cells within the rhizosphere.     

Perturbation of host ubiquitin systems by plant pathogen/pest effector proteins

Microbial pathogens and pests of animals and plants secrete effector proteins into host cells, altering cellular physiology to the benefit of the invading parasite. Research in the past decade has delivered significant new insights into the molecular mechanisms of how these effector proteins function, with a particular focus on modulation of host immunity‐related pathways. One host system that has emerged as a common target of effectors is the ubiquitination system in which substrate proteins are post‐translationally modified by covalent conjugation with the small protein ubiquitin. This modification, typically via isopeptide bond formation through a lysine side chain of ubiquitin, can result in target degradation, relocalization, altered activity or affect protein–protein interactions. In this review, I focus primarily on how effector proteins from bacterial and filamentous pathogens of plants and pests perturb host ubiquitination pathways that ultimately include the 26S proteasome. The activities of these effectors, in how they affect ubiquitin pathways in plants, reveal how pathogens have evolved to identify and exploit weaknesses in this system that deliver increased pathogen fitness.     

The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea

BACKGROUND: Plants have evolved efficient mechanisms to combat pathogen attack. One of the earliest responses to attempted pathogen attack is the generation of oxidative burst that can trigger hypersensitive cell death. This is called the hypersensitive response (HR) and is considered to be a major element of plant disease resistance. The HR is thought to deprive the pathogens of a supply of food and confine them to initial infection site. Necrotrophic pathogens, such as the fungi Botrytis cinerea and Sclerotinia sclerotiorum, however, can utilize dead tissue. RESULTS: Inoculation of B. cinerea induced an oxidative burst and hypersensitive cell death in Arabidopsis. The degree of B. cinerea and S. sclerotiorum pathogenicity was directly dependent on the level of generation and accumulation of superoxide or hydrogen peroxide. Plant cells exhibited markers of HR death, such as nuclear condensation and induction of the HR-specific gene HSR203J. Growth of B. cinerea was suppressed in the HR-deficient mutant dnd1, and enhanced by HR caused by simultaneous infection with an avirulent strain of the bacterium Pseudomonas syringae. HR had an opposite (inhibitory) effect on a virulent (biotrophic) strain of P. syringae. Moreover, H(2)O(2) levels during HR correlated positively with B. cinerea growth but negatively with growth of virulent P. syringae. CONCLUSIONS: We show that, although hypersensitive cell death is efficient against biotrophic pathogens, it does not protect plants against infection by the necrotrophic pathogens B. cinerea and S. sclerotiorum. By contrast, B. cinerea triggers HR, which facilitates its colonization of plants. Hence, these fungi can exploit a host defense mechanism for their pathogenicity.     

Regulatory network modelling of iron acquisition by a fungal pathogen in contact with epithelial cells

Background  

Reverse engineering of gene regulatory networks can be used to predict regulatory interactions of an organism faced with environmental changes, but can prove problematic, especially when focusing on complicated multi-factorial processes. Candida albicans is a major human fungal pathogen. During the infection process, this fungus is able to adapt to conditions of very low iron availability. Such adaptation is an important virulence attribute of virtually all pathogenic microbes. Understanding the regulation of iron acquisition genes will extend our knowledge of the complex regulatory changes during the infection process and might identify new potential drug targets. Thus, there is a need for efficient modelling approaches predicting key regulatory events of iron acquisition genes during the infection process.     

The targeting of plant cellular systems by injected type III effector proteins

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector–host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.     

Biotechnological potential of engineering pathogen effector proteins for use in plant disease management

A key component in the management of many diseases of crops is the use of plant disease resistance genes. However, the discovery and then sequence identification of these plant genes is challenging, whereas the characterization of the molecules that they recognize, the effector/avirulence products in pathogens, is often considerably more straight forward. Effectors are small proteins secreted by pathogens that can play major roles in modulating a plant's defense against attack. Effectors can be used to guide breeding of resistance genes, to trigger defense responses, and are part of integrated disease management strategies for crop protection. This review covers the role of effector-driven biotechnology in controlling plant diseases caused by fungi or oomycetes. Given that multi-billion dollar agriculture crops are based in some cases on plants recognizing just a handful of such effector proteins, there is considerable scope to use more fully effector proteins as a biotechnology resource in agriculture.     

Phenotypic plasticity facilitates initial colonization of a novel environment

Phenotypic plasticity can allow organisms to respond to environmental changes by producing better matching phenotypes without any genetic change. Because of this, plasticity is predicted to be a major mechanism by which a population can survive the initial stage of colonizing a novel environment. We tested this prediction by challenging wild Drosophila melanogaster with increasingly extreme larval environments and then examining expression of alcohol dehydrogenase (ADH) and its relationship to larval survival in the first generation of encountering a novel environment. We found that most families responded in the adaptive direction of increased ADH activity in higher alcohol environments and families with higher plasticity were also more likely to survive in the highest alcohol environment. Thus, plasticity of ADH activity was positively selected in the most extreme environment and was a key trait influencing fitness. Furthermore, there was significant heritability of ADH plasticity that can allow plasticity to evolve in subsequent generations after initial colonization. The adaptive value of plasticity, however, was only evident in the most extreme environment and had little impact on fitness in less extreme environments. The results provide one of the first direct tests of the adaptive role of phenotypic plasticity in colonizing a novel environment.     

Bacterial effector interplay: a new way to view effector function

Bacterial pathogens are dependent on virulence factors to efficiently colonize and propagate within their hosts. Many Gram-negative bacterial pathogens rely on specialized proteinaceous secretion systems that inject virulence factors, termed effectors, directly into host cells. These bacterial effector proteins perform various functions within host cells; however, regulation of their function within the host cell is highly enigmatic. It is becoming increasingly apparent that many of these effectors directly influence and regulate each other and their mechanisms within the host cell. We discuss the emerging theme of bacterial effector interplay impacting infection and the importance of investigating this topic.     

Bacterial toxin and effector glycosyltransferases

Clostridial glucosylating cytotoxins, including Clostridium difficile toxins A and B, Clostridium novyi α-toxin, and Clostridium sordellii lethal toxin, are major virulence factors and causative agents of human diseases. These toxins mono-O-glucosylate (or mono-O-GlcNAcylate) a specific threonine residue of Rho/Ras-proteins, which is essential for the function of the molecular switches. Recently, a related group of glucosyltransferases from Legionella pneumophila has been identified. These Legionella glucosyltransferases modify the large GTPase elongation factor eEF1A at a serine residue by mono-O-glucosylation, thereby inhibiting protein synthesis of target cells. Recent results on structures, functions and biological roles of both groups of bacterial toxin glucosyltransferases will be discussed.     

Plasmopara viticola effector PvRXLR131 suppresses plant immunity by targeting plant receptor-like kinase inhibitor BKI1

The grapevine downy mildew pathogen Plasmopara viticola secretes a set of RXLR effectors (PvRXLRs) to overcome host immunity and facilitate infection, but how these effectors function is unclear. Here, the biological function of PvRXLR131 was investigated via heterologous expression. Constitutive expression of PvRXLR131 in Colletotrichum gloeosporioides significantly enhanced its pathogenicity on grapevine leaves. Constitutive expression of PvRXLR131 in Arabidopsis promoted Pseudomonas syringae DC3000 and P. syringae DC3000 (hrcC^-) growth as well as suppressed defence-related callose deposition. Transient expression of PvRXLR131 in Nicotiana benthamiana leaves could also suppress different elicitor-triggered cell death and inhibit plant resistance to Phytophthora capsici. Further analysis revealed that PvRXLR131 interacted with host Vitis vinifera BRI1 kinase inhibitor 1 (VvBKI1), and its homologues in N. benthamiana (NbBKI1) and Arabidopsis (AtBKI1). Moreover, bimolecular fluorescence complementation analysis revealed that PvRXLR131 interacted with VvBKI1 in the plasma membrane. Deletion assays showed that the C-terminus of PvRXLR131 was responsible for the interaction and mutation assays showed that phosphorylation of a conserved tyrosine residue in BKI1s disrupted the interaction. BKI1 was a receptor inhibitor of growth- and defence-related brassinosteroid (BR) and ERECTA (ER) signalling. When silencing of NbBKI1 in N. benthamiana, the virulence function of PvRXLR131 was eliminated, demonstrating that the effector activity is mediated by BKI1. Moreover, PvRXLR131-transgenic plants displayed BKI1-overexpression dwarf phenotypes and suppressed BR and ER signalling. These physiological and genetic data clearly demonstrate that BKI1 is a virulence target of PvRXLR131. We propose that P. viticola secretes PvRXLR131 to target BKI1 as a strategy for promoting infection.     

Type III effector proteins from the plant pathogen Xanthomonas and their role in the interaction with the host plant

Pathogenicity of Xanthomonas campestris pathovar (pv.) vesicatoria and most other Gram-negative bacterial plant pathogens largely depends on a type III secretion (TTS) system which is encoded by hypersensitive response and pathogenicity (hrp) genes. These genes are induced in the plant and are essential for the bacterium to be virulent in susceptible hosts and for the induction of the hypersensitive response (HR) in resistant host and non-host plants. The TTS machinery secretes proteins into the extracellular milieu and effector proteins into the plant cell cytosol. In the plant, the effectors presumably interfere with cellular processes to the benefit of the pathogen or have an avirulence activity that betrays the bacterium to the plant surveillance system. Type III effectors were identified by their avirulence activity, co-regulation with the TTS system and homology to known effectors. A number of effector proteins are members of families, e.g., the AvrBs3 family in Xanthomonas. AvrBs3 localizes to the nucleus of the plant cell where it modulates plant gene expression. Another family that is also present in Xanthomonas is the YopJ/AvrRxv family. The latter proteins appear to act as SUMO cysteine proteases in the host. Here, we will present an overview about the regulation of the TTS system and its substrates and discuss the function of the AvrRxv and AvrBs3 family members in more detail.     

Background matching behaviour and pathogen acquisition: plant site preference does not predict the bacterial acquisition efficiency of vectors

Many insect-borne pathogens are heterogeneously distributed within their hosts: therefore, a vector’s within-plant distribution may be a predictor of its exposure to pathogens. In this study, we set out to quantify plant site preference, in the context of background matching, and investigated its effect on acquisition of a bacterial pathogen by its leafhopper vectors. The two green-coloured species, Graphocephala atropunctata and Draeculacephala minerva, preferred green plant tissue and artificial backgrounds whereas the brown-coloured Homalodisca vitripennis preferred brown stem tissue and backgrounds. Within-plant feeding site did not predict either the acquisition success or the number of plant-pathogenic Xylella fastidiosa cells acquired by the vectors; an 86% mortality for G. atropunctata was reported on the lignified stem tissue. Overall, H. vitripennis acquired significantly more cells than G. atropunctata. A novel artificial diet-based transmission system was used to further illustrate that the observed between-species difference in the number of cells acquired was independent of vector-host plant interactions. H. vitripennis, a less efficient vector of the bacterium X. fastidiosa on grapevines, acquired more bacterial cells than G. atropunctata, possibly due to its larger size. Contrary to previous assumptions, pathogen acquisition efficiency by the vectors did not explain their reported differences in inoculation. Vector interactions with the host during the inoculation stage should be evaluated as another determinant of X. fastidiosa transmission efficiency.     

Bacterial genomics and pathogen evolution

The availability of hundreds of bacterial genome sequences has altered the study of bacterial pathogenesis, affecting both design of experiments and analysis of results. Comparative genomics and genomic tools have been used to identify virulence factors and genes involved in environmental persistence of pathogens. However, a major stumbling block in the genomics revolution has been the large number of genes with unknown function that have been identified in every organism sequenced to date.     

Bacterial colonization of a fumigated alkaline saline soil

After chloroform fumigating an arable soil, the relative abundance of phylotypes belonging to only two phyla (Actinobacteria and Firmicutes) and two orders [Actinomycetales and Bacillales (mostly Bacillus)] increased in a subsequent aerobic incubation, while it decreased for a wide range of bacterial groups. It remained to be seen if similar bacterial groups were affected when an extreme alkaline saline soil was fumigated. Soil with electrolytic conductivity between 139 and 157 dS m^?1, and pH 10.0 and 10.3 was fumigated and the bacterial community structure determined after 0, 1, 5 and 10 days by analysis of the 16S rRNA gene, while an unfumigated soil served as control. The relative abundance of the Firmicutes increased in the fumigated soil (52.8 %) compared to the unfumigated soil (34.2 %), while that of the Bacteroidetes decreased from 16.2 % in the unfumigated soil to 8.8 % in the fumigated soil. Fumigation increased the relative abundance of the genus Bacillus from 14.7 % in the unfumigated soil to 25.7 %. It was found that phylotypes belonging to the Firmicutes, mostly of the genus Bacillus, were dominant in colonizing the fumigated alkaline saline as found in the arable soil, while the relative abundance of a wide range of bacterial groups decreased.