The AAP gene family for amino acid permeases contributes to development of the cyst nematode Heterodera schachtii in roots of Arabidopsis

The beet cyst nematode Heterodera schachtii is able to infect Arabidopsis plants and induce feeding sites in the root. These syncytia are the only source of nutrients for the nematodes throughout their life and are a nutrient sink for the host plant. We have studied here the role of amino acid transporters for nematode development. Arabidopsis contains a large number of different amino acid transporters in several gene families but those of the AAP family were found to be especially expressed in syncytia. Arabidopsis contains 8 AAP genes and they were all strongly expressed in syncytia with the exception of AAP5 and AAP7, which were slightly downregulated. We used promoter::GUS lines and in situ RT-PCR to confirm the expression of several AAP genes and LHT1, a lysine- and histidine-specific amino acid transporter, in syncytia. The strong expression of AAP genes in syncytia indicated that these transporters are important for the transport of amino acids into syncytia and we used T-DNA mutants for several AAP genes to test for their influence on nematode development. We found that mutants of AAP1, AAP2, and AAP8 significantly reduced the number of female nematodes developing on these plants. Our study showed that amino acid transport into syncytia is important for the development of the nematodes

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

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

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

Inhibition of acetyl-CoA carboxylase by spirotetramat causes growth arrest and lipid depletion in nematodes

Plant-parasitic nematodes pose a significant threat to agriculture causing annual yield losses worth more than 100 billion US$. Nematode control often involves the use of nematicides, but many of them including non-selective fumigants have been phased out, particularly due to ecotoxicological concerns. Thus new control strategies are urgently needed. Spirotetramat (SPT) is used as phloem-mobile systemic insecticide targeting acetyl-CoA carboxylase (ACC) of pest insects and mites upon foliar application. However, in nematodes the mode of action of SPT and its effect on their development have not been studied so far. Our studies revealed that SPT known to be activated in planta to SPT-enol acts as a developmental inhibitor of the free-living nematode Caenorhabditis elegans and the plant-parasitic nematode Heterodera schachtii. Exposure to SPT-enol leads to larval arrest and disruption of the life cycle. Furthermore, SPT-enol inhibits nematode ACC activity, affects storage lipids and fatty acid composition. Silencing of H. schachtii ACC by RNAi induced similar phenotypes and thus mimics the effects of SPT-enol, supporting the conclusion that SPT-enol acts on nematodes by inhibiting ACC. Our studies demonstrated that the inhibition of de novo lipid biosynthesis by interfering with nematode ACC is a new nematicidal mode of action addressed by SPT, a well-known systemic insecticide for sucking pest control

Identification of Two Meloidogyne hapla Genes and an Investigation of Their Roles in the Plant-Nematode Interaction

Root-knot nematodes are soil-borne pathogens that invade and establish feeding sites in plant roots. They have an extremely broad host range, including most vascular plants. During infection of a susceptible host, root-knot nematodes secrete molecules called effectors that help them establish an intimate interaction with the plant and, at the same time, allow them to evade or suppress plant immune responses. Despite the fact that Meloidogyne hapla is a significant pest on several food crops, no effectors have been characterized from this root-knot nematode species thus far. Using the published genome and proteome from M. hapla, we have identified and characterized two genes, MhTTL2 and Mh265. MhTTL2 encodes a predicted secreted protein containing a transthyretin-like protein domain. The expression of MhTTL2 was up-regulated during parasitic life stages of the nematode, and in situ hybridization showed that MhTTL2 was expressed in the amphids, suggesting it has a role in the nematode nervous system during parasitism. We also studied the gene Mh265. The Mh265 transcript was localized to the subventral esophageal glands. An upregulation in Mh265 expression coincided with the pre- and early-parasitic life stages of the nematode. When Mh265 was constitutively expressed in plants, it enhanced their susceptibility to nematodes. These transgenic plants were also compromised in flg22-induced callose deposition, suggesting the Mh265 is modulating plant basal immune responses

Heterodera schachtii Tyrosinase-like protein - a novel nematode effector modulating plant hormone homeostasis

The beet cyst nematode Heterodera schachtii causes major yield losses in sugar beet. Understanding the interaction between H. schachtii and its host plant is important for developing a sustainable management system. Nematode effectors play a crucial role in initializing and sustaining successful parasitism. In our study, we identified a gene (Hs-Tyr) encoding a tyrosinase functional domain (PF00264). We describe Hs-Tyr as a novel nematode effector. Hs-Tyr is localized in the nematode esophageal gland. Up-regulation of its expression coincided with the parasitic developmental stages of the nematode. Silencing Hs-Tyr by RNA interference made the treated nematodes less virulent. When RNAi-treated nematodes succeeded in infecting the plant, developing females and their associated syncytial nurse cells were significantly smaller than in control plants. Ectopically expressing the Hs-Tyr effector in Arabidopsis increased plant susceptibility to H. schachtii, but not to the root-knot nematode Meloidogyne incognita. Interestingly, Hs-Tyr in the plant promoted plant growth and changed the root architecture. Additionally, the expression of Hs-Tyr in Arabidopsis caused changes in the homeostasis of several plant hormones especially auxin and the ethylene precursor aminocyclopropane-carboxylic acid