Arabidopsis HIPP27 is a host susceptibility gene for the beet cyst nematode Heterodera schachtii

Sedentary plant‐parasitic cyst nematodes are obligate biotrophs that infect the roots of their host plant. Their parasitism is based on the modification of root cells to form a hypermetabolic syncytium from which the nematodes draw their nutrients. The aim of this study was to identify nematode susceptibility genes in Arabidopsis thaliana and to characterize their roles in supporting the parasitism of Heterodera schachtii. By selecting genes that were most strongly upregulated in response to cyst nematode infection, we identified HIPP27 (HEAVY METAL‐ASSOCIATED ISOPRENYLATED PLANT PROTEIN 27) as a host susceptibility factor required for beet cyst nematode infection and development. Detailed expression analysis revealed that HIPP27 is a cytoplasmic protein and that HIPP27 is strongly expressed in leaves, young roots and nematode‐induced syncytia. Loss‐of‐function Arabidopsis hipp27 mutants exhibited severely reduced susceptibility to H. schachtii and abnormal starch accumulation in syncytial and peridermal plastids. Our results suggest that HIPP27 is a susceptibility gene in Arabidopsis whose loss of function reduces plant susceptibility to cyst nematode infection without increasing the susceptibility to other pathogens or negatively affecting the plant phenotype.     

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.     

Gene expression profiling and shared promoter motif for cell wall-modifying proteins expressed in soybean cyst nematode-infected roots

We hypothesized that soybean cyst nematode (SCN; Heterodera glycines) co-opts part or all of one or more innate developmental process in soybean (Glycine max) to establish its feeding structure, syncytium, in soybean roots. The syncytium is formed within the vascular bundle by partial degradation of cell walls and membranes between adjacent parenchyma cells. A mature syncytium incorporates as many as 200 cells into one large multinucleated cell. Gene expression patterns for several cell wall-modifying proteins were compared in multiple tissues undergoing major shifts in cell wall integrity. These included SCN-colonized roots, root tips where vascular differentiation occurs, flooded roots (aerenchyma), adventitious rooting in hypocotyls, and leaf abscission zones. A search in the 5' upstream promoters of these genes identified a motif (SCNbox1: WGCATGTG) common to several genes that were up-regulated in several different tissues. The polygalacturonase 11 promoters (GmPG11a/b) include the SCNbox1 motif. The expression pattern for GmPG11a was examined further in transgenic soybean containing a PG11a promoter fused to a β-glucuronidase (GUS) reporter gene. GUS expression was highest in cells undergoing radial expansion in the stele and/or cell wall dissolution. GUS staining was not observed in cortical cells where a lateral root tip or a growing nematode emerged through the root cortex.     

Sucrose supply to nematode-induced syncytia depends on the apoplasmic and symplasmic pathways

The plant parasitic nematode Heterodera schachtii induces syncytial feeding structures in the roots of host plants. Nematode-induced syncytia become strong sink tissues in the plant solute circulation system as the parasites start withdrawing nutrients. In the present work, the expression pattern of the phloem-specific sucrose transporter AtSUC4 (also described as AtSUT4) is analysed in syncytia induced by H. schachtii and it is compared with that of AtSUC2, another phloem-specific sucrose transporter, which is expressed in syncytia. The temporal expression pattern was monitored by GUS-tests and real-time RT-PCR, while the localization within the syncytia was performed using in situ RT-PCR. In this context, the concentration of sucrose in infection sites was also analysed and, in fact, an increase in response to syncytium development was found. Silencing of the AtSUC4 gene finally resulted in a significant reduction of female nematode development, thus demonstrating a function for this gene for the first time. It is therefore concluded that AtSUC4 plays a significant role in the early phase of syncytium differentiation when functional plasmodesmata to the phloem are not yet established. It is further concluded that, during syncytium establishment, transporters are responsible for sucrose supply and, at a later stage, when a connection to the phloem is established via plasmodesmata, transporters are required for sucrose retrieval.     

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.     

An Arabidopsis ATPase gene involved in nematode‐induced syncytium development and abiotic stress responses

The beet cyst nematode Heterodera schachtii induces syncytia in the roots of Arabidopsis thaliana, which are its only nutrient source. One gene, At1g64110, that is strongly up‐regulated in syncytia as shown by RT‐PCR, quantitative RT‐PCR, in situ RT‐PCR and promoter::GUS lines, encodes an AAA+‐type ATPase. Expression of two related genes in syncytia, At4g28000 and At5g52882, was not detected or not different from control root segments. Using amiRNA lines and T‐DNA mutants, we show that At1g64110 is important for syncytium and nematode development. At1g64110 was also inducible by wounding, jasmonic acid, salicylic acid, heat and cold, as well as drought, sodium chloride, abscisic acid and mannitol, indicating involvement of this gene in abiotic stress responses. We confirmed this using two T‐DNA mutants that were more sensitive to abscisic acid and sodium chloride during seed germination and root growth. These mutants also developed significantly smaller roots in response to abscisic acid and sodium chloride. An in silico analysis showed that ATPase At1g64110 (and also At4g28000 and At5g52882) belong to the ‘meiotic clade’ of AAA proteins that includes proteins such as Vps4, katanin, spastin and MSP1.     

Changes in the structure ofArabidopsis thaliana during female development of the plant-parasitic nematodeHeterodera schachtii

Summary The beet cyst nematodeHeterodera schachtii is able to establish a feeding structure (syncytium) in the vascular tissue of roots and shoots ofArabidopsis thaliana. Histological and ultrastructural studies were performed to assess plant responses during the development of juvenile females under monoxenic conditions. After destructively invading a root the nematode selects and pierces a single procambial cell with its stylet and transforms it into an initial syncytial cell (ISC) by secretory activity. The first most obvious changes in the ISC occur in the vacuolar system and at the wall. Differentiation of a central vacuole is impeded resulting in the formation of numerous small vacuoles. Multivesicular and paramural bodies are formed. An electron translucent material is deposited on the cell wall. Partial dissolution of the cell wall leads to the formation of a syncytium. At the juveniles' last pre-adult developmental stage the syncytium attains its maximum longitudinal and radial extension, occupying a major part of the central cylinder. Its features are indicative of a very high level of metabolic activity. The hypertrophied syncytium is ensheathed by a peridermal cover in which secondary xylem and phloem elements are interspersed. When females die the syncytia degenerate. The ultrastructural and histological features of syncytia described from roots are also found in syncytia induced in aerial parts of the plant.