Delivery of macromolecules to plant parasitic nematodes using a tobacco rattle virus vector

Plant parasitic nematodes cause significant damage to crops on a worldwide scale. These nematodes are often soil dwelling but rely on plants for food and to sustain them during reproduction. Complex interactions occur between plants and nematodes during the nematode life cycle with plant roots developing specialized feeding structures through which nematodes withdraw nutrients. Here we describe a novel method for delivering macromolecules to feeding nematodes using a virus-based vector [tobacco rattle virus (TRV)]. We show that the parasitic nematode Heterodera schachtii will ingest fluorescent proteins transiently expressed in plant roots infected with a TRV construct carrying the appropriate protein sequence. A prerequisite for this delivery is the presence of replicating virus in root tips prior to the formation of nematode-induced syncytia. We show also that TRV vectors expressing nematode gene sequences can be used to induce RNAi in the feeding nematodes.     

Induction of phloem unloading in Arabidopsis thaliana roots by the parasitic nematode Heterodera schachtii.

Phloem unloading of both the fluorescent probe carboxyfluorescein (CF) and 14C-labeled solutes was induced in Arabidopsis thaliana L. roots by the parasitic nematode Heterodera schachtii Schmidt. Confocal laser scanning microscopy demonstrated that anomalous unloading of CF from the sieve element companion cell complexes occurred specifically into the syncytium, the nematode-induced feeding structure located within the stele of the root. From this syncytial complex of modified root cells, both fluorescent and radioactive labels were withdrawn by feeding nematodes. Movement of CF was unidirectional from the phloem to the syncytium. A range of low-molecular-weight fluorescent probes (including CF) microinjected into the syncytium stayed in this structure, demonstrating that it is symplastically isolated from the surrounding root tissue. The mechanism of unloading in this host-pathogen relationship therefore appears to be apoplastic. Our results provide unequivocal evidence that sedentary cyst-forming nematodes have direct access to phloem-derived solutes.     

The Arabidopsis thaliana Sucrose Transporter Gene AtSUC4 is Expressed in Meloidogyne incognita-induced Root Galls

The plant parasitic nematode Meloidogyne incognita is as an obligate parasite entirely dependent on the plants solute supply. Therefore, the nematodes induce the formation of several giant cells which are embedded into root galls. At present only little information is available about the solute transfer mechanisms of the plants to supply the induced galls and giant cells and consequently the nematodes. In the present work we could show by phloem-loading experiments that giant cells in the roots of Arabidopsis thaliana are not symplasmically connected to the phloem elements, thus differing considerably form the comparable plant–nematode interaction of Arabidopsis and Heterodera schachtii . Consequently the gene expression of the sucrose transporter AtSUC4 ( AtSUT4 ) was studied during nematode development, and its functionality was shown using RNAi gene silencing lines.     

Syncytia formed by adult female Heterodera schachtii in Arabidopsis thaliana roots have a distinct cell wall molecular architecture

? Plant-parasitic cyst nematodes form a feeding site, termed a syncytium, through which the nematode obtains nutrients from the host plant to support nematode development. The structural features of cell walls of syncytial cells have yet to be elucidated. ? Monoclonal antibodies to defined glycans and a cellulose-binding module were used to determine the cell wall architectures of syncytial and surrounding cells in the roots of Arabidopsis thaliana infected with the cyst nematode Heterodera schachtii. ? Fluorescence imaging revealed that the cell walls of syncytia contain cellulose and the hemicelluloses xyloglucan and heteromannan. Heavily methyl-esterified pectic homogalacturonan and arabinan are abundant in syncytial cell walls; galactan could not be detected. This is suggestive of highly flexible syncytial cell walls. ? This work provides important information on the structural architecture of the cell walls of this novel cell type and reveals factors that enable the feeding site to perform its functional requirements to support nematode development.     

The interaction of the novel 30C02 cyst nematode effector protein with a plant β-1,3-endoglucanase may suppress host defence to promote parasitism

Phytoparasitic nematodes secrete an array of effector proteins to modify selected recipient plant cells into elaborate and essential feeding sites. The biological function of the novel 30C02 effector protein of the soybean cyst nematode, Heterodera glycines, was studied using Arabidopsis thaliana as host and the beet cyst nematode, Heterodera schachtii, which contains a homologue of the 30C02 gene. Expression of Hg30C02 in Arabidopsis did not affect plant growth and development but increased plant susceptibility to infection by H. schachtii. The 30C02 protein interacted with a specific (AT4G16260) host plant β-1,3-endoglucanase in both yeast and plant cells, possibly to interfere with its role as a plant pathogenesis-related protein. Interestingly, the peak expression of 30C02 in the nematode and peak expression of At4g16260 in plant roots coincided at around 3-5 d after root infection by the nematode, after which the relative expression of At4g16260 declined significantly. An Arabidopsis At4g16260 T-DNA mutant showed increased susceptibility to cyst nematode infection, and plants that overexpressed At4g16260 were reduced in nematode susceptibility, suggesting a potential role of host β-1,3-endoglucanase in the defence response against H. schachtii infection. Arabidopsis plants that expressed dsRNA and its processed small interfering RNA complementary to the Hg30C02 sequence were not phenotypically different from non-transformed plants, but they exhibited a strong RNA interference-mediated resistance to infection by H. schachtii. The collective results suggest that, as with other pathogens, active suppression of host defence is a critical component for successful parasitism by nematodes and a vulnerable target to disrupt the parasitic cycle.     

Nematodes as vectors to introduce Agrobacterium into plant roots

A fast plant promoter test was developed by means of a nematode to transfer Agrobacterium tumefaciens into plant roots. Two-week-old Arabidopsis thaliana (L.) Heynh. plants were transferred to infection medium. Meloidogyne incognita or Heterodera schachtii juveniles were mixed with the Agrobacterium strain that harboured the binary vector, and this mixture was used for plant inoculation. During migration of the nematode and establishment of the feeding site inside the roots, the T-DNA was delivered into the root cells. A few days later, the infected plants could be analysed for expression of the T-DNA reporter gene in and around the nematode feeding sites (NFS), without the need to go first through the whole transformation and regeneration procedure. Depending on the construct, expression of the β-glucuronidase gene in the NFS or along the migration path of the nematode could be seen in the roots of Arabidopsis plants. Furthermore, stably transformed plants could be regenerated from the infected roots.     

AtCDKA;1 silencing in Arabidopsis thaliana reduces reproduction of sedentary plant-parasitic nematodes

SUMMARY: The activity of the Arabidopsis thaliana cyclin-dependent kinase AtCDKA;1 is important throughout G(1)/S and G(2)/M transitions and guarantees the progression of the cell cycle. Inhibitor studies have shown that activation of the cell cycle is important for the development of nematode feeding sites. The aim of this study was to silence the expression of the AtCDKA;1 gene in nematode feeding sites to interfere with their development. Therefore, sense and antisense constructs were made for the AtCDKA;1 gene and fused to a nematode-inducible promoter which was activated in nematode feeding sites at an earlier time point than AtCDKA;1. Two transgenic A. thaliana lines (S266 and S306) containing inverted repeats of the AtCDKA;1 gene and with reduced AtCDKA;1 expression in seedlings and galls were analysed in more detail. When the lines were infected with the root-knot nematode Meloidogyne incognita, significantly fewer galls and egg masses developed on the roots of the transgenic than wild-type plants. Infection of the AtCDKA;1-silenced lines with Heterodera schachtii resulted in significantly fewer cysts compared with controls. The S266 and S306 lines showed no phenotypic aberrations in root morphology, and analysis at different time points after infection demonstrated that the number of penetrating nematodes was the same, but fewer nematodes developed to maturity in the silenced lines. In conclusion, our results demonstrate that silencing of CDKA;1 can be used as a strategy to produce transgenic plants less susceptible to plant-parasitic nematodes.     

Use of real-time quantitative PCR to investigate root and gall colonisation by co-inoculated isolates of the nematophagous fungus Pochonia chlamydosporia

The fungus Pochonia chlamydosporia is a potential biological control agent for plant parasitic nematodes, but to date, there has been little investigation of interactions (competitive, antagonistic or synergistic) between different isolates that occur together on roots and nematode galls. Real-time quantitative PCR (qPCR) has greatly improved the study of many fungi in situ on plant and nematode hosts, but distinguishing closely related isolates remains difficult. In this study, primers to discriminate P. chlamydosporia var. chlamydosporia and P. chlamydosporia var. catenulata were used to measure the relative abundance of isolates of the two varieties when inoculated singly or together on tomato plants. Also, sequence-characterised amplified polymorphic regions were identified to distinguish two different isolates of P. chlamydosporia var. chlamydosporia . Individual 1-cm root segments and nematode galls were excised, DNA extracted and subjected to real-time qPCR with the discriminatory primers. The qPCR method proved sensitive and reproducible and demonstrated that roots and nematode galls were not uniformly colonised by the fungi. Results indicated that the P. chalmydosporia var. catenulata isolate was more abundant on roots and eggs than P. chlamydosporia var. chlamydosporia , but all the isolates infected a similar proportion of nematode eggs. There was an indication that the abundance of each fungal isolate was reduced in co-inoculation experiments compared with single inoculations, but the number of root segments and galls colonised was not statistically significantly different.