Elżbieta Różańska

Parasitic Worms Stimulate Host NADPH Oxidases to Produce Reactive Oxygen Species That Limit Plant Cell Death and Promote Infection

Reactive oxygen species restrict cell death at sites of parasitic nematode infection and support nurse cell formation in plant roots. Promoting Parasitism with ROS Some species of nematode worms can invade the roots of plants and establish a feeding site composed of a large syncytial plant cell. This biotrophic lifestyle requires that the worms find a way to suppress plants’ immune responses. One aspect of plant immunity is the production of reactive oxygen species (ROS) that damage pathogens and promote plant cell death to limit the spread of infection. Siddique et al. found that deleting the enzymes that produce ROS in Arabidopsis thaliana plants responding to infection by Heterodera schachtii worms prevented the worms from establishing syncytia and growing within roots, suggesting that the worms have co-opted plant ROS as a means of promoting parasitism. Thus, plant ROS can play both positive and negative roles during infection. Plants and animals produce reactive oxygen species (ROS) in response to infection. In plants, ROS not only activate defense responses and promote cell death to limit the spread of pathogens but also restrict the amount of cell death in response to pathogen recognition. Plants also use hormones, such as salicylic acid, to mediate immune responses to infection. However, there are long-lasting biotrophic plant-pathogen interactions, such as the interaction between parasitic nematodes and plant roots during which defense responses are suppressed and root cells are reorganized to specific nurse cell systems. In plants, ROS are primarily generated by plasma membrane–localized NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, and loss of NADPH oxidase activity compromises immune responses and cell death. We found that infection of Arabidopsis thaliana by the parasitic nematode Heterodera schachtii activated the NADPH oxidases RbohD and RbohF to produce ROS, which was necessary to restrict infected plant cell death and promote nurse cell formation. RbohD- and RbohF-deficient plants exhibited larger regions of cell death in response to nematode infection, and nurse cell formation was greatly reduced. Genetic disruption of SID2, which is required for salicylic acid accumulation and immune activation in nematode-infected plants, led to the increased size of nematodes in RbohD- and RbohF-deficient plants, but did not decrease plant cell death. Thus, by stimulating NADPH oxidase–generated ROS, parasitic nematodes fine-tune the pattern of plant cell death during the destructive root invasion and may antagonize salicylic acid–induced defense responses during biotrophic life stages.

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants

Significance Sedentary plant-parasitic cyst nematodes are microscopic roundworms that cause significant yield losses in agriculture. Successful parasitism is based on the formation of a hypermetabolic feeding site in host roots from which the nematodes withdraw their nutrients. The host cell cycle is activated at the site of infection and contributes to the formation of the syncytium. Here, we provide genetic evidence that nematode-derived cytokinin is involved in activating the host cell cycle during infection. Our findings show the ability of an animal to synthesize and secrete a functional plant hormone to establish long-term parasitism. Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

Piriformospora indica antagonizes cyst nematode infection and development in Arabidopsis roots

The beneficial endophytic fungus Piriformospora indica colonizes the roots of many plant species, including the model plant Arabidopsis thaliana. Its colonization promotes plant growth, development, and seed production as well as resistance to various biotic and abiotic stresses. In the present work, P. indica was tested as potential antagonist of the sedentary plant-parasitic nematode Heterodera schachtii. This biotrophic cyst-forming nematode induces severe host plant damage by changing the morphogenesis and physiology of infected roots. Here it is shown that P. indica colonization, as well as the application of fungal exudates and cell-wall extracts, significantly affects the vitality, infectivity, development, and reproduction of H. schachtii.

Systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of Arabidopsis thaliana infected with the beet cyst nematode Heterodera schachtii : Photosynthesis and ROS in nematode-infected plants

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second-stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post-inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down-regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter Rfd , defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non-photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine-tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes.

The role of the secondary phloem during the development of the grapevine Berry Shrivel ripening disorder

Berry Shrivel (BS) is a post-veraison physiological ripening disorder of grapevine berries. Its symptoms encompass low pH, reduced content of sugars and anthocyanins, and loss of turgor leading to berries shriveling. Evidence for the primary causes of BS is still speculative and anatomical studies are scarce. So far, anatomical studies have determined necrotic cells, degraded primary phloem cells and hardening of secondary phloem cells in the rachis of BS affected grapes. The picture is far from being complete. Herein we report in-depth analyses of the ultrastructure, anatomy and spatial elementary analysis of rachis and pedicel tissues of BS symptomatic grape clusters with different symptom severity. We hypothesize that structural changes in the vascular system of BS affected grape clusters could alter transport functions of the phloem tissue and contribute to the appearance of BS symptoms. By applying different microscopic techniques (LM, SEM, TEM and EDS) we found a number of anatomical differences in both, rachis and pedicels, between H and BS symptomatic grapes, which include: (i) extended areas of collapsed cells and cell wall thickenings in the secondary phloem in BS samples; (ii) reduced number of cell layers in the cambium in BS samples; (iii) higher rate of callose deposition on sieve plates that are additionally covered with a carbohydrate-like material in BS samples; and (iv) reduced (up to 60%) estimated sieve tube conductivity in BS samples.

Host factors influence the sex of nematodes parasitizing roots of Arabidopsis thaliana

Plant-parasitic cyst nematodes induce hypermetabolic syncytial nurse cells in the roots of their host plants. Syncytia are their only food source. Cyst nematodes are sexually dimorphic, with their differentiation into male or female strongly influenced by host environmental conditions. Under favorable conditions with plenty of nutrients, more females develop, whereas mainly male nematodes develop under adverse conditions such as in resistant plants. Here, we developed and validated a method to predict the sex of beet cyst nematode (Heterodera schachtii) during the early stages of its parasitism in the host plant Arabidopsis thaliana. We collected root segments containing male-associated syncytia (MAS) or female-associated syncytia (FAS), isolated syncytial cells by laser microdissection, and performed a comparative transcriptome analysis. Genes belonging to categories of defense, nutrient deficiency, and nutrient starvation were overrepresented in MAS as compared to FAS. Conversely, gene categories related to metabolism, modification, and biosynthesis of cell walls were overrepresented in FAS. We used β-glucuronidase (GUS) analysis, qRT-PCR, and loss-of-function mutants to characterize FAS- and MAS-specific candidate genes. Our results demonstrated that various plant-based factors, including immune response, nutrient availability, and structural modifications, influence the sexual fate sex determination of cyst the nematodes.

Heavy metal tolerance in contrasting ecotypes of Alyssum montanum

The response of metallicolous (M) and nonmetallicolous (NM) Alyssum montanum ecotypes to multi-metal stress was investigated under in vitro condition and compared in this study. Shoot cultures were simultaneously treated with 0.7 mM ZnSO4, 3.0 μM Pb(NO3)2 and 16.4 μM CdCl2 for 8 weeks and evaluated for their morphogenetic and ultrastructural reaction, growth tolerance as well as ability to Zn, Pb, and Cd uptake. Moreover, tissue localization and concentrations of antioxidant compounds were determined in order to elucidate the potential role of ROS-scavenging machinery in plant tolerance to metal toxicity. The results clearly demonstrated that M specimens treated with heavy metals showed less phytotoxic symptoms and low level of lipid peroxidation than reference NM one. The enhanced tolerance of M ecotype resulted from heavy metals detoxification in trichomes and intracellular leaf compartments as well as balanced ROS accumulation. The inactivation of ROS in M plants was based on peroxidase-flavonoid system, while in NM plants such relationship was not detected and amounts of antioxidant enzymes or phenolic compounds was comparable to untreated specimens or decreased significantly. Considering the procumbent growth of such hemicryptophyte which reproduce effectively in the presence of heavy metals but is characterized by low biomass production, it is proposed to exploit M ecotype of A. montanum in revegetation schemes of polluted calamine wastes to provide the prompt stabilization of areas prone to erosion.

Genetics of lineage diversification and the evolution of host usage in the economically important wheat curl mite, Aceria tosichella Keifer, 1969

BackgroundUnderstanding the mechanisms that underlie the diversification of herbivores through interactions with their hosts is important for their diversity assessment and identification of expansion events, particularly in a human-altered world where evolutionary processes can be exacerbated. We studied patterns of host usage and genetic structure in the wheat curl mite complex (WCM), Aceria tosichella, a major pest of the world’s grain industry, to identify the factors behind its extensive diversification.ResultsWe expanded on previous phylogenetic research, demonstrating deep lineage diversification within the taxon, a complex of distinctive host specialist and generalist lineages more diverse than previously assumed. Time-calibrated phylogenetic reconstruction inferred from mitochondrial DNA sequence data suggests that lineage diversification pre-dates the influence of agricultural practices, and lineages started to radiate in the mid Miocene when major radiations of C4 grasses is known to have occurred. Furthermore, we demonstrated that host specificity is not phylogenetically constrained, while host generalization appears to be a more derived trait coinciding with the expansion of the world’s grasslands. Demographic history of specialist lineages have been more stable when compared to generalists, and their expansion pre-dated all generalist lineages. The lack of host-associated genetic structure of generalists indicates gene flow between mite populations from different hosts.ConclusionsOur analyses demonstrated that WCM is an unexpectedly diverse complex of genetic lineages and its differentiation is likely associated with the time of diversification and expansion of its hosts. Signatures of demographic histories and expansion of generalists are consistent with the observed proliferation of the globally most common lineages. The apparent lack of constrains on host use, coupled with a high colonization potential, hinders mite management, which may be further compromised by host range expansion. This study provides a significant contribution to the growing literature on host-association and diversification in herbivorous invertebrates.