Miroslaw Sobczak

Characterization of susceptibility and resistance responses to potato cyst nematode (Globodera spp.) infection of tomato lines in the absence and presence of the broad-spectrum nematode resistance Hero gene

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.

Formation of wall openings in root cells of Arabidopsis thaliana following infection by the plant-parasitic nematode Heterodera schachtii

The induction and differentiation of feeding structures (syncytia) of the cyst nematode Heterodera schachtii in roots of Arabidopsis thaliana is accompanied by drastic cellular modifications. We investigated the formation of cell wall openings which occurred during syncytium differentiation. At the beginning of syncytium induction, a callose-like layer was deposited inside of the wall of the initial syncytial cell (ISC). First wall dissolutions developed by gradual widening of plasmodesmata between the ISC and neighbouring cells. As a general thickening of syncytial cell walls blocked existing plasmodesmata, other large openings were formed by enzymatic dissolution of intact walls by putative cellulase activity.

Changes in the structure of Arabidopsis thaliana roots induced during development of males of the plant parasitic nematode Heterodera schachtii

Plant parasitic nematodes of the genus Heterodera show a high degree of sexual dimorphism, which is reflected by different nutritional demands and differences in the structure of the induced specific syncytial feeding site in the plant. The determination of the sex of the nematode Heterodera schachtii and other related species was repeatedly reported to be dependent on trophic factors, which are provided by the induced syncytia. The structural differences of syncytia induced by H. schachtii in roots of Arabidopsis thaliana were analysed at the anatomical and ultrastructural level. Syncytia of males were induced in the root pericycle. The developing syncytium then expanded into procambial or cambial cells of the vascular cylinder. Differentiated vascular elements were not included. The expansion of the syncytium triggered the proliferation of cambial and peridermal tissues, in a manner similar to secondary growth, and the formation of additional xylem and phloem elements. In comparison to syncytia associated with females, syncytia associated with males were less hypertrophied and were composed of more cells. Distinct cell wall openings were mostly found between the few strongly hypertrophied syncytial elements at the actual feeding site in the pericycle. The ultrastructure was very similar to female-associated syncytia but showed conspicuous differences in the structure and localization of cell wall ingrowths. These ingrowths were rare and weakly developed and occurred not only at the interface with xylem elements but also at the internal and external walls of the syncytia. After feeding had ceased at the end of the third developmental stage the syncytia degenerated.

Localization of hydrogen peroxide during the defence response of Arabidopsis thaliana against the plant-parasitic nematode Heterodera glycines

Hydrogen peroxide (H 2 O 2 ) production during the infection of Arabidopsis thaliana by the soybean cyst nematode Heterodera glycines was detected histochemically by the reaction of H 2 O 2 with cerium chloride producing four different patterns of electron-dense precipitates of cerium perhydroxides. As A. thaliana is not a regular host of H. glycines , the defence response is considerable, but does not completely inhibit the development of the nematode. H 2 O 2 was produced not only by cells mechanically damaged during invasion and feeding site induction by the nematode, but also by cells surrounding developing syncytia and cells which were neither in contact with the nematode nor with the syncytium. Die Lokalisation von Peroxid wahrend der Abwehrreaktion von Arabidopsis thaliana gegen den pflanzenparasitaren Nematoden Heterodera glycines - Die Bildung von Wasserstoffperoxid (H 2 O 2 ) im Rahmen der Infektion von Arabidopsis thaliana durch den Sojabohnen-Zystennematoden Heterodera glycines wurde histochemisch durch die Reaktion von H 2 O 2 mit Cerchlorid nachgewiesen, wobei vier verschiedene Muster elektronendichter Prazipitate von Cerperhydroxiden gebildet wurden. Da A. thaliana kein regularer Wirt von H. glycine s ist, kommt es zu einer betrachtlichen Abwehrreaktion, die jedoch die Entwicklung des Nematoden nicht vollstandig verhindert. H 2 O 2 wurde nicht nur von Zellen produziert, die im Laufe des Eindringens und der Induktion des Nahrzellensystems durch den Nematoden mechanisch beschadigt worden waren, sondern auch von Zellen, die sich entwickelnde Syncytien umgaben und von Zellen, die weder mit dem Nematoden noch mit dem Syncytium in Kontakt standen.

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.

The Structure of Syncytia

Infective second stage juveniles of the cyst forming genera of plant parasitic nematodes (Heterodera and Globodera) induce the formation of feeding sites in roots of resistant and susceptible plants. Under special experimental conditions in Arabidopsis thaliana, future males select the initial syncytial cell (ISC) in the pericycle and future females select their ISC in the procambium. After a preparation phase the nematode feeding site is still unicellular. Cytoplasmic streaming and cytoplasm density, amount of endoplasmic reticulum (ER) and volume of nucleus are increased while the volume of central vacuole is decreased and new small cytoplasmic vacuoles are formed. The cell walls are thickened and covered by a layer of callose-like material. The developing syncytia expand along the vascular cylinder. The expansion of the syncytium triggers the proliferation of cambial and peridermal tissues, in a manner similar to secondary growth. Compared with syncytia associated with females, syncytia of males are less hypertrophied and are composed of more cells. Distinctive cell wall openings are mostly found between the few strongly hypertrophied pericyclic syncytial elements. The ultrastructure of both types of syncytia is very similar but shows conspicuous differences in the structure and localisation of cell wall ingrowths.

The plant cell wall in the feeding sites of cyst nematodes.

Plant parasitic cyst nematodes (genera Heterodera and Globodera) are serious pests for many crops. They enter the host roots as migratory second stage juveniles (J2) and migrate intracellularly toward the vascular cylinder using their stylet and a set of cell wall degrading enzymes produced in the pharyngeal glands. They select an initial syncytial cell (ISC) within the vascular cylinder or inner cortex layers to induce the formation of a multicellular feeding site called a syncytium, which is the only source of nutrients for the parasite during its entire life. A syncytium can consist of more than hundred cells whose protoplasts are fused together through local cell wall dissolutions. While the nematode produces a cocktail of cell wall degrading and modifying enzymes during migration through the root, the cell wall degradations occurring during syncytium development are due to the plants own cell wall modifying and degrading proteins. The outer syncytial cell wall thickens to withstand the increasing osmotic pressure inside the syncytium. Furthermore, pronounced cell wall ingrowths can be formed on the outer syncytial wall at the interface with xylem vessels. They increase the surface of the symplast-apoplast interface, thus enhancing nutrient uptake into the syncytium. Processes of cell wall degradation, synthesis and modification in the syncytium are facilitated by a variety of plant proteins and enzymes including expansins, glucanases, pectate lyases and cellulose synthases, which are produced inside the syncytium or in cells surrounding the syncytium.

Starch Serves as Carbohydrate Storage in Nematode-Induced Syncytia

The plant parasitic nematode Heterodera schachtii induces specific syncytial feeding sites in the roots of Arabidopsis thaliana from where it withdraws all required nutrients. Therefore, syncytia have to be well supplied with assimilates and generate strong sinks in the host plants transport system. Import mechanisms and consequent accumulation of sucrose in syncytia were described recently. In this work, we studied the starch metabolism of syncytia. Using high-performance liquid chromatography and microscopic analyses, we demonstrated that syncytia store carbohydrates by starch accumulation. Further, we monitored the expression of genes involved in the starch metabolic pathway by gene chip analysis and quantitative reverse transcription-PCR. Finally, we provide functional proof of the importance of starch synthesis for nematode development using T-DNA insertion lines. We conclude that syncytia accumulate starch as a carbohydrate buffer to compensate for changing solute uptake by the nematode and as long-term storage during juvenile development.

Ultrastructure of feeding plugs and feeding tubes formed by Heterodera schachtii

The development of feeding plugs and feeding tubes formed in syncytia induced by the cyst-forming nematode Heterodera schachtii in roots of Arabidopsis thaliana was examined at the ultrastructural level. The feeding plug was first observed 24 h after selection of the initial syncytial cell (ISC) and was present throughout the entire nematode life cycle. In later stages of nematode development the feeding plug became increasingly robust and infiltrated by fibrillar syncytial wall material while the central part, through which the nematode stylet was inserted, retained an amorphous structure. Neither the feeding plug nor the nematode stylet were observed to penetrate the plasmalemma of the syncytium. After the nematode completed the preparation phase for feeding, the first secretions were released from the stylet orifice and emitted through the plasmalemma into the cytoplasm. They formed uniformly osmiophilic wavy tubes without an electron translucent lumen. The first typical feeding tubes were found 24 h after ISC selection and were composed of an electron dense wall and an electron translucent lumen. The size of a single feeding tube was about 1 X 4 mum. No difference occurred between feeding tubes formed by male and female juveniles. Frequently, membranes of the endoplasmic reticulum were connected to the wall of feeding tubes. After the nematode completed feeding, the tubes were disassociated from the stylet orifice and were dispersed in the syncytial cytoplasm. The feeding tube lumen was filled with cytoplasm and the wall gradually degraded. Die Ultrastruktur der von Heterodera schachtii gebildeten Dichtungsstopfen und Saugrohrchen - Die Entwicklung von Dichtungsstopfen (feeding plugs) und Saugrohrchen (feeding tubes), die in den von dem Zystennematoden Heterodera schachtii induzierten Synzytien in den Wurzeln von Arabidopsis thaliana gebildet werden, wurden auf der Ebene der Ultrastruktur untersucht. Die ersten Dichtungsstopfen wurden 24 h nach der Auswahl der Synzytiuminitialzelle (ISC) durch den Nematoden beobachtet. Sie waren wahrend des gesamten Lebenszyklus des Nematoden vorhanden. In den spateren Entwicklungsstadien der Nematoden wurde der Dichtungsstopfen zunehmend robuster und von fiblillarem Material aus der Zellwand durchdrungen. Der zentrale Teil des Stopfens, durch den der Mundstachel des Nematoden in die ISC ragte, behielt eine amorphe Struktur. Es wurde nicht beobachtet, dass der Dichtungsstopfen oder der Mundstachel das Plasmalemma durchbrachen. Nachdem der Nematode die vorbereitende Phase der Nahrungsaufnahme abgeschlossen hatte, traten die ersten Sekrete aus der Mundstacheloffnung aus und wurden durch das Plasmalemma hindurch in das Zytoplasma abgegeben. Sie bildeten gleichmassig osmiophile, wellige Rohrchen ohne ein elektronendurchlassiges Lumen. Die ersten typischen Saugrohrchen wurden 24 h nach der Auswahl der ISC gefunden. Sie bestanden aus einer elektronendichten Wand und einem elektronendurchlassigen Lumen. Die Grosse eines einzelnen Saugrohrchens betrug ungefahr 1 X 4 mum. Zwischen den von mannlichen oder von weiblichen Juvenilen gebildeten Saugrohrchen traten keine Unterschiede auf. Haufig waren Membranen des endoplasmatischen Reticulums mit den Wanden von Saugrohrchen verbunden. Wenn der Nematode die Nahrungsaufnahme abgeschlossen hatte, wurden die Saugrohrchen von der Offnung des Mundstachels getrennt und im synzytialen Zytoplama verteilt. Das Lumen dieser Saugrohrchen war mit Zytoplasma gefullt, und die Wand wurde langsam abgebaut.