Identification and characterization of effector proteins of the beet cyst nematode <em>Heterodera schachtii</em>

Plant parasitic nematodes (PPN) are considered as economically important pests of a wide range of plants including ornamentals, vegetables, and fruit trees. The beet cyst nematode Heterodera schachtii causes massive yield loss in sugar beet production. H. schachtii is a biotrophic sedentary endoparasite which depends on a specific hypermetabolic syncytial nurse cell structure in the host root. To induce and maintain the feeding site, H. schachtii uses a specific set of effector proteins that are secreted mostly from the oesophageal glands. Identification and functional analysis of these proteins are crucial steps to understand the nature of nematode parasitism. In our study, we sequenced the H. schachtii transcriptome via Illumina MiSeq. We compared the assembled H. schachtii transcriptome with the available nematode ESTs from NEMBASE4 and transcripts from available nematode transcriptomes in order to identify new effectors. We found 484 putative secretory proteins specific to plant-parasitic nematodes (PSP). Further comparison with known H. schachtii ESTs resulted in the identification of so far unknown PSPs. The annotation of the identified PSPs showed enrichment in certain gene ontologies such as metabolic and catalytic activities in addition to growth regulation function. We selected two genes for a detailed functional analysis. Through Pfam domain analysis, we identified “HsPDI” encoding a protein disulfide-isomerase domain and “Hs-Tyr” encoding a tyrosinase functional domain. To our knowledge, these two domains have not yet been described in the context of nematode effector proteins. Transcripts of both genes were localized in the esophageal gland of pre-parasitic juveniles, and their expression was found to be up-regulated during the parasitic developmental stages. Silencing of both genes by RNAi affected nematode development and syncytium formation: both females and syncytia were significantly smaller than the controls. On the contrary, ectopic expression of the effectors in Arabidopsis increased plant susceptibility to H. schachtii. Silencing of HsPDI led to syncytia with distinct ultrastructural changes such as less dense cytoplasm with distorted and degraded organelles. Treating HsPDI-expressing Arabidopsis plants with the defense inducing peptide flg22 triggered ROS burst, but the measured H2O2 level was lower compared with control plants. Furthermore, treating pre-infective nematode juveniles with H2O2 caused up-regulation of HsPDI expression. Silencing HsPDI in pre-infective nematode juveniles induced higher sensitivity to H2O2 stress compared with untreated nematodes. Fluorescence microscopy of Nicotiana benthamiana leaves transiently expressing HsPDI::GFP showed that it is specifically located in the apoplastic space. Thus, our results demonstrate the importance of the HsPDI for the interaction between nematode and host as an apoplastic effector, and indicates the possible function of HsPDI as a scavenger of plant ROS. Ectopic expression of Hs-Tyr in Arabidopsis has a clear impact on plant growth: shoot growth was promoted and root architecture was changed. No changes where observed in the root length or weight. Additionally, the presence of Hs-Tyr in the plant caused changes in the homeostasis of several plant hormones especially auxin, jasmonate precursor cisOPDA and the ethylene precursor ACC. No significant changes of jasmonic acid and salicylic acid levels were observed. The transgenic plants were more susceptible to H. schachtii, but not to the root-knot nematode Meloidogyne incognita. This indicates that this effector is of specific importance for the parasitism of the cyst nematode H. schachtii. The results suggest that Hs-Tyr interferes with the orchestration of plant hormones in a still unknown way. The presented results show that the analyzed PSPs have specific effects on nematode-plant interaction and therefore serve as effectors facilitating parasitism.Pflanzenparasitäre Nematoden sind wirtschaftlich bedeutsame Schaderreger an vielen landwirtschaftlichen Kulturen wie auch Zierpflanzen, Gemüse- und Obstarten. Der Rübenzystennematode Heterodera schachtii verursacht massive Ertragsverluste in der Zuckerrübenproduktion. H. schachtii ist ein biotropher, sedentärer Endoparasit, dessen Entwicklung von der Bildung eines hypermetabolischen Nährzellensystems in der Wurzel abhängt. Um dieses Nährzellensystem zu induzieren und auf Dauer zu erhalten, verfügt H. schachtii über Effektorproteine, die überwiegend in den Ösophagusdrüsen gebildet werden. Die Identifizierung und funktionelle Analyse dieser Effektoren sind wichtige Schritte auf dem Weg zu einem tieferen Verständnis des Parasitismus des Nematoden. Für unsere Analysen sequenzierten wir das Transkriptom von H. schachtii mit einem Illumina MiSeq Gerät. Anschließend wurde das zusammengesetzte Transkriptom mit den verfügbaren ESTs aus der Datenbank NEMABASE4 und Transkriptomen weiterer Nematoden verglichen, um neue Effektorseqzenzen zu identifizieren. Auf diese Weise wurden zunächst 484 mutmaßlich sekretierte Proteine (PSP) identifiziert. Eine weiterer Sequenzvergleich mit bekannten ESTs von H. schachtii führte zur Identifizierung bisher unbekannter PSPs. Die Annotation dieser neuen PSPs ergab eine Anreicherung bestimmter Genontologien wie z.B. metabolischen und katalytischen Aktivitäten und wachstumsregulierenden Funktionen. Es wurden zwei Gene für eine detaillierte funktionelle Analyse ausgewählt. Mit Hilfe einer Pfam-Domänenanalyse konnten “HsPDI”, das für eine protein disulfide-isomerase Domäne, sowie “Hs-Tyr”, das für eine Tyrosinase-Domäne kodiert, identifiziert werden. Beide Domänen waren bisher noch nicht in Zusammenhang mit Nematoden-Effektoren gebracht worden. Die Transkripte beider Gene konnten in den Ösophagusdrüsen von prä-infektiösen Nematodenlarven lokalisiert werden, wobei die Genexpression während der parasitischen Entwicklungsstadien aufreguliert ist. Das Stilllegen der beiden Gene mit Hilfe von RNAi hatte Auswirkungen auf die Nematodenentwicklung wie auch auf die Bildung des Nährzellensystems: sowohl Weibchen als auch Nährzellen waren kleiner als in unbehandelten Kontrollen. Im Gegensatz dazu führte die ektopische Expression der Gene in Arabidopsispflanzen zu einer erhöhten Anfälligkeit gegenüber H. schachtii. Die Stilllegung von HsPDI wirkte sich auf die Ultrastruktur der gebildeten Nährzellen in Form eines weniger dichten Zytoplasmas mit veränderten und degradierten Organellen aus. Wurden transgene Arabidopsispflanzen, die HsPDI exprimierten, mit dem Abwehr induzierenden Peptid flg22 behandelt, so reagierten diese mit starker ROS-Produktion, die H2O2-Konzentration war jedoch geringer als in nicht transgenen Kontrollpflanzen. Die Stilllegung von HsPDI in prä-infektösen Nematodenlarven führte zu deren erhöhter Sensitivität gegenüber einer Behandlung mit H2O2 im Vergleich zu einer Kontrollgruppe. Die fluoreszenzmikroskopische Analyse von Blättern von Nicotiana benthamiana, die HsPDI::GFP transient exprimierten, zeigte, dass HsPDI im Apoplasten lokalisiert ist. Die Ergebnisse der durchgeführten Experimente zeigen, dass HsPDI ein Effektor ist, der eine wichtige Rolle in der Interaktion zwischen Nematode und Wirtspflanze spielt, wobei es vermutlich als Radikalfänger für ROS pflanzlicher Herkunft fungiert. Die ektopische Expression von Hs-Tyr in Arabidopsis hatte eine deutliche Auswirkung auf das Pflanzenwachstum: Das Sprosswachtum war verstärkt, die Wurzelarchitektur war verändert, wobei die Wurzellänge und das Wurzelgewicht unverändert blieben. Die transgenen Pflanzen wiesen darüber hinaus deutliche Veränderung der Homöostase verschiedener Hormone, vor allem Auxin, dem Jasmonatvorläufer cisOPDA und dem Ethylenvorläufer ACC auf. Die Konzentrationen von Jasmonsäure und Salizylsäure waren dagegen gleich bleibend. Die transgenen Pflanzen waren gegenüber H. schachtii anfälliger, nicht jedoch gegenüber dem Wurzelgallennematoden Meloidogyne incognita. Daraus läßt sich schließen, dass der Effektor physiologische Prozesse in Gang setzt, die spezifisch für den Parasitismus des Zystennematoden H. schachtii von Bedeutung sind. Die bisherigen Ergebnisse weisen darauf hin, dass Hs-Tyr dabei in einer noch unbekannten Weise in den Hormonhaushalt der Pflanze eingreift. Die gewonnenen Erkenntnisse zeigen, dass die untersuchten PSPs spezifische Effekte auf die Interaktion zwischen Nematode und Pflanze haben und somit als Effektoren den Parasitismus des Nematoden unterstützen

Signatures of adaptation to a monocot host in the plant-parasitic cyst nematode Heterodera sacchari.

Interactions between plant-parasitic nematodes and their hosts are mediated by effectors, i.e. secreted proteins that manipulate the plant to the benefit of the pathogen. To understand the role of effectors in host adaptation in nematodes, we analysed the transcriptome of Heterodera sacchari, a cyst nematode parasite of rice (Oryza sativa) and sugarcane (Saccharum officinarum). A multi-gene phylogenetic analysis showed that H. sacchari and the cereal cyst nematode Heterodera avenae share a common evolutionary origin and that they evolved to parasitise monocot plants from a common dicot-parasitic ancestor. We compared the effector repertoires of H. sacchari with those of the dicot parasites Heterodera glycines and Globodera rostochiensis to understand the consequences of this transition. While, in general, effector repertoires are similar between the species, comparing effectors and non-effectors of H. sacchari and G. rostochiensis shows that effectors have accumulated more mutations than non-effectors. Although most effectors show conserved spatiotemporal expression profiles and likely function, some H. sacchari effectors are adapted to monocots. This is exemplified by the plant-peptide hormone mimics, the CLAVATA3/EMBRYO SURROUNDING REGION-like (CLE) effectors. Peptide hormones encoded by H. sacchari CLE effectors are more similar to those from rice than those from other plants, or those from other plant-parasitic nematodes. We experimentally validated the functional significance of these observations by demonstrating that CLE peptides encoded by H. sacchari induce a short root phenotype in rice, whereas those from a related dicot parasite do not. These data provide a functional example of effector evolution that co-occurred with the transition from a dicot-parasitic to a monocot-parasitic lifestyle.BBSR

Identification and characterization of a putative protein disulfide isomerase (HsPDI) as an alleged effector of Heterodera schachtii

The plant-parasitic nematode Heterodera schachtii is an obligate biotroph that induces syncytial feeding sites in roots of its hosts. Nematodes produce effectors that are secreted into the host and facilitate infection process. Here we identified H. schachtii protein disulphide isomerase (HsPDI) as a putative effector that interferes with the host's redox status. In situ hybridization showed that HsPdi is specifically localized within esophageal glands of pre-parasitic second stage juveniles (J2). HsPdi is up-regulated in the early parasitic J2s. Silencing of HsPdi by RNA interference in the J2s hampers their development and leads to structural malfunctions in associated feeding sites induced in Arabidopsis roots. Expression of HsPDI in Arabidopsis increases plant's susceptibility towards H. schachtii. HsPdi expression is up-regulated in the presence of exogenous H2O2, whereas HsPdi silencing results in increased mortality under H2O2 stress. Stable expression of HsPDI in Arabidopsis plants decreases ROS burst induced by flg22. Transiently expressed HsPDI in N. benthamiana leaves is localized in the apoplast. HsPDI plays an important role in the interaction between nematode and plant, probably through inducing local changes in the redox status of infected host tissue. It also contributes to protect the nematode from exogenous H2O2 stress

In vitro life cycle of Heterodera sacchari on Pluronic gel

This paper presents studies on the life cycle of Heterodera sacchari under in vitro conditions. Pluronic gel was used as a medium for growth of H. sacchari. The life cycle was completed in 7-9 weeks on rice (Oryza sativa, 'Nipponbare'). After infection, juveniles developed and reached the reproducing adult female stage at 25 days post inoculation (dpi). At 35 dpi, all females produced eggs in various numbers. Some females were translucent and eggs inside could be counted. At 49 dpi females started to tan and developed into dark brown cysts. Hatching of H. sacchari juveniles from cysts could be stimulated by 3 mM ZnCl2 but not by rice root exudates. The in vitro culture of H. sacchari on Pluronic gel can be used efficiently to collect post-infective nematode/host samples at different time points for various studies and to screen different rice cultivars for resistance/susceptibility.</p

In vitro life cycle of Heterodera sacchari on Pluronic gel

This paper presents studies on the life cycle of Heterodera sacchari under in vitro conditions. Pluronic gel was used as a medium for growth of H. sacchari. The life cycle was completed in 7-9 weeks on rice (Oryza sativa, 'Nipponbare'). After infection, juveniles developed and reached the reproducing adult female stage at 25 days post inoculation (dpi). At 35 dpi, all females produced eggs in various numbers. Some females were translucent and eggs inside could be counted. At 49 dpi females started to tan and developed into dark brown cysts. Hatching of H. sacchari juveniles from cysts could be stimulated by 3 mM ZnCl2 but not by rice root exudates. The in vitro culture of H. sacchari on Pluronic gel can be used efficiently to collect post-infective nematode/host samples at different time points for various studies and to screen different rice cultivars for resistance/susceptibility.</p