Differential gene expression in Arabidopsis following infection by plant-parasitic nematodes Meloidogyne incognita and Heterodera schachtii

Whole genome microarrays were used to study plant gene expression in mature Meloidogyne incognita -induced galls in Arabidopsis. We found 959 genes to be significantly differentially expressed, and two-thirds of these were down-regulated. Microarray results were confirmed by qRT-PCR. The temporal and spatial responses of four differentially expressed genes were analysed using GUS reporter plants following infection with M. incognita and the cyst nematode Heterodera schachtii . The ammonium transporter gene AtAMT1;2 was consistently and locally repressed in response to both nematodes at all developmental stages. The lateral organ boundary domain gene LBD41 showed up-regulation in the feeding sites of both nematode species, although there was variation in expression in saccate H. schachtii female feeding sites. Expression of an actin depolymerizing factor ADF3 and a lipid transfer protein was induced in feeding sites of both nematodes at the fusiform stage and this persisted in feeding sites of saccate M. incognita . These results contribute to the knowledge of how plant gene expression responds to parasitism by these nematodes as well as highlighting further differences in the mechanisms of development and maintenance of these feeding site structures.     

Regulatory sequences of Arabidopsis drive reporter gene expression in nematode feeding structures.

In the quest for plant regulatory sequences capable of driving nematode-triggered effector gene expression in feeding structures, we show that promoter tagging is a valuable tool. A large collection of transgenic Arabidopsis plants was generated. They were transformed with a beta-glucuronidase gene functioning as a promoter tag. Three T-DNA constructs, pGV1047, p delta gusBin19, and pMOG553, were used. Early responses to nematode invasion were of primary interest. Six lines exhibiting beta-glucuronidase activity in syncytia induced by the beet cyst nematode were studied. Reporter gene activation was also identified in galls induced by root knot and ectoparasitic nematodes. Time-course studies revealed that all six tags were differentially activated during the development of the feeding structure. T-DNA-flanking regions responsible for the observed responses after nematode infection were isolated and characterized for promoter activity.     

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.     

The tobacco Cel7 gene promoter is auxin-responsive and locally induced in nematode feeding sites of heterologous plants

Emerging evidence suggests that plant cell-wall-modifying enzymes induced by root-parasitic nematodes play important roles in feeding cell formation. We previously identified a tobacco endo-β-1,4-glucanase (cellulase) gene, NtCel7 , that was strongly induced in both root-knot and cyst nematode feeding cells. To characterize further the developmental and nematode-responsive regulation of NtCel7 , we isolated the NtCel7 promoter and analysed its expression over a time course of nematode infection and in response to auxin, gibberellin, ethylene and sucrose in soybean and tomato hairy roots and in Arabidopsis containing the NtCel7 promoter fused to the β-glucuronidase (GUS) reporter gene. Histochemical analyses of transgenic plant materials revealed that the NtCel7 promoter exhibited a unique organ-specific expression pattern during plant development suggestive of important roles for NtCel7 in both vegetative and reproductive growth. In all plant species tested, strong GUS expression was observed in root tips and lateral root primordia of uninfected roots with weaker expression in the root vasculature. Further analyses of transgenic Arabidopsis plants revealed expression in shoot and root meristems and the vasculature of most organs during plant development. We also determined that the NtCel7 promoter was induced by auxin, but not gibberellin, ethylene or sucrose. Moreover, strong GUS activity was observed in both cyst and root-knot nematode-induced feeding sites in transgenic roots of soybean, tomato and Arabidopsis. The conserved developmental and nematode-responsive expression of the NtCel7 promoter in heterologous plants indicates that motifs of this regulatory element play a fundamental role in regulating NtCel7 gene expression within nematode feeding sites and that this regulation may be mediated by auxin.     

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.     

rha1, a gene encoding a small GTP binding protein from Arabidopsis, is expressed primarily in developing guard cells.

The rha1 gene from Arabidopsis encodes a small GTP binding protein belonging to the Ypt/Rab family. Transgenic Arabidopsis plants containing the promoter region of the rha1 gene fused to the beta-glucuronidase (gus) reporter gene revealed gus expression limited mainly to the guard cells of stomata, the stipules, and the root tip of young plants. In flowering plants, expression was found predominantly in the receptacle and in guard cells of the different flower organs. High GUS activity could also be seen in callus tissue and developing seeds. No detectable activity was present in other plant tissues; activity could not be induced by various treatments. GUS activity was visualized histochemically using both 5-bromo-4-chloro-3-indolyl beta-D-glucuronide and a newly developed GUS substrate: Sudan II-beta-glucuronide. The latter precipitates as red crystals at the site of GUS activity. Results obtained by the gus analysis were confirmed by whole-mount mRNA in situ hybridization. A hypothesis for the function of the Rha1 protein is discussed.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

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.     

Genetic analysis and epigenetic silencing of At4CL1 and At4CL2 expression in transgenic Arabidopsis

4-coumarate::CoA ligase (4CL) gene family members are involved in channeling carbon flow into branch pathways of phenylpropanoid metabolism. Transgenic Arabidopsis plants containing the At4CL1 or At4CL2 promoter fused to the beta-glucuronidase (GUS) reporter gene show developmentally regulated GUS expression in the xylem tissues of the root and shoot. To identify regulatory genes involved in the developmental regulation of At4CL and other phenylpropanoid-specific genes, we generated ethyl methyl sulfate mutagenized populations of At4CL1::GUS and At4CL2::GUS transgenic lines and screened approximately 16,000 progeny for reduced or altered GUS expression. Several lines with reproducible patterns of reduced GUS expression were identified. However, the GUS-expression phenotype segregated in a non-Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5-azacytidine (aza) treatment, suggesting inhibitory DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation of At4CL::GUS lines with further At4CL promoter constructs enhanced the GUS-silencing phenotype. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two different modes of silencing were engaged in the At4CL1::GUS and At4CL2::GUS silenced lines. While silencing in the seedlings of the At4CL1::GUS lines was root specific in seedlings, it affected all organs in the At4CL2::GUS lines. Also, At4CL1::GUS transgene silencing was confined to the transgene but At4CL2::GUS silencing extended to the endogenous At4CL2 gene. Organ-specific silencing of the At4CL1::GUS transgene cannot be explained by current models in the literature.     

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.     

The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia

The Arabidopsis thaliana MERISTEM LAYER 1 (ATML1) gene is expressed in the epidermis of developing embryos and shoot meristems. To identify regulatory sequences necessary for epidermis-specific expression, three fusions of overlapping ATML1 genomic sequences to the GUS reporter gene were introduced into Arabidopsis plants. All fusion genes conferred epidermis-specific expression of both GUS mRNA and protein activity but varied in both the timing and relative levels of expression, suggesting partial redundancy of ATML1 regulatory elements. This study defines L1-specific regulatory sequences that are sufficient to direct foreign gene expression in a layer-specific manner.     

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.