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.     

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.     

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.     

Evidence of differences between the communities of arbuscular mycorrhizal fungi colonizing galls and roots of Prunus persica infected by the root-knot nematode Meloidogyne incognita

Arbuscular mycorrhizal fungi (AMF) play important roles as plant protection agents, reducing or suppressing nematode colonization. However, it has never been investigated whether the galls produced in roots by nematode infection are colonized by AMF. This study tested whether galls produced by Meloidogyne incognita infection in Prunus persica roots are colonized by AMF. We also determined the changes in AMF composition and biodiversity mediated by infection with this root-knot nematode. DNA from galls and roots of plants infected by M. incognita and from roots of noninfected plants was extracted, amplified, cloned, and sequenced using AMF-specific primers. Phylogenetic analysis using the small-subunit (SSU) ribosomal DNA (rDNA) data set revealed 22 different AMF sequence types (17 Glomus sequence types, 3 Paraglomus sequence types, 1 Scutellospora sequence type, and 1 Acaulospora sequence type). The highest AMF diversity was found in uninfected roots, followed by infected roots and galls. This study indicates that the galls produced in P. persica roots due to infection with M. incognita were colonized extensively by a community of AMF, belonging to the families Paraglomeraceae and Glomeraceae, that was different from the community detected in roots. Although the function of the AMF in the galls is still unknown, we hypothesize that they act as protection agents against opportunistic pathogens.     

Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis

? Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide. ? The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita. ? Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression. ? The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.     

The Importance of the Host Plant on the Interaction Between Root-knot Nematodes Meloidogyne spp. and the Nematophagous Fungus, Verticillium chlamydosporium Goddard

The effect of the host plant on the efficacy of Verticillium chlamydosporium as a biological control agent for root-knot nematodes was investigated in four experiments. The growth of the fungus in the rhizosphere differed significantly with different plant species, the brassicas kale and cabbage supporting the most extensive colonization. The presence of nematodes in roots increased the growth of the fungus on most plants, and this effect was associated with the emergence of egg masses on the root surface; the presence of Meloidogyne incognita did not stimulate growth of the fungus in the rhizosphere until 5 weeks after the addition of infective juveniles to soil. The susceptibility of the plant host to M. incognita attack influenced the numbers of nematode eggs parasitized by the fungus. The control of the nematode was less effective on tomato roots, which produced large galls as a result of nematode infection compared with control on potato roots where galls were smaller, despite the greater abundance of the fungus in the rhizosphere of tomato plants. In large galls, a significant proportion of the egg masses remained embedded in the roots and was isolated from the fungus which was confined to the rhizosphere. Hence, the plant species has a marked effect on the efficacy of V. chlamydosporium as a biological control agent.     

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.     

TcYSL3, a member of the YSL gene family from the hyper-accumulator Thlaspi caerulescens, encodes a nicotianamine-Ni/Fe transporter

The two main features of plant hyper-accumulator species are the massive translocation of heavy metal ions to the aerial parts and their tolerance to such high metal concentrations. Recently, several lines of evidence have indicated a role for nicotianamine (NA) in metal homeostasis, through the chelation and transport of NA-metal complexes. The function of transport of NA-metal chelates, required for the loading and unloading of vessels, has been assigned to the Yellow Stripe 1 (YSL)-Like family of proteins. We have characterized three YSL genes in Thlaspi caerulescens in the context of hyper-accumulation. The three YSL genes are expressed at high rates compared with their Arabidopsis thaliana homologs but with distinct patterns. While TcYSL7 was highly expressed in the flowers, TcYSL5 was more highly expressed in the shoots, and the expression of TcYSL3 was equivalent in all the organs tested. In situ hybridizations have shown that TcYSL7 and TcYSL5 are expressed around the vasculature of the shoots and in the central cylinder in the roots. The exposure to heavy metals (Zn, Cd, Ni) does not affect the high and constitutive expression of the TcYSL genes. Finally, we have demonstrated by mutant yeast complementation and uptake measurements that TcYSL3 is an Fe/Ni-NA influx transporter. This work provides therefore molecular, histological and biochemical evidence supporting a role for YSL transporters in the overall scheme of NA and NA-metal, particularly NA-Ni, circulation in a metal hyper-accumulator plant.     

Inheritance of resistance to Meloidogyne incognita race 2 in the hot pepper cultivar Carolina Cayenne (Capsicum annuum L.)

Root-knot nematodes of the genus Meloidogyne are important pathogens affecting vegetable crop production in Brazil and worldwide. The pepper species Capsicum annuum includes both hot and sweet peppers; very little emphasis has been placed on breeding sweet peppers for nematode resistance. We report on the inheritance of resistance to Meloidogyne incognita (Kofoid & White) Chitwood race 2 in the hot pepper cultivar Carolina Cayenne. The hot pepper cv. Carolina Cayenne was used as seed parent and the sweet pepper cv. Agron?mico-8 was used as pollen parent to obtain the F(1) and F(2) generations and the backcross generations BC(11) and BC(12). The plants were inoculated with M. incognita race 2 at a rate of 60 eggs/ml of substrate and, after a suitable incubation period, the numbers of root galls and egg masses per root system were evaluated on each plant. Broad- (0.77 and 0.72) and narrow-sense (0.77 and 0.63) heritability estimates were high for both root galls and egg masses, respectively. The mean degree of dominance was estimated as 0.29 and 0.25 for numbers of galls and egg masses, respectively; these estimates were not significantly different from 0, indicating a predominantly additive gene action. The results were consistent with a hypothesis of monogenic resistance in Carolina Cayenne.     

Molecular cloning and chromosomal mapping of type one serine/threonine protein phosphatases in Arabidopsis thaliana

Type one serine/threonine protein phosphatases (PP1s) have been implicated in various processes of plant growth and development. In all plant species studied, PP1s are encoded by multigene families. Previous studies in our laboratory identified five Arabidopsis thaliana PP1 genes (TOPP1, TOPP2, TOPP3, TOPP4 and TOPP5). In the present study, we report the isolation of three additional PP1 genes (TOPP6, TOPP7 and TOPP8). Southern blot analyses indicate that these three newly isolated genes are single-copy genes in A. thaliana genome. All the three genes are expressed in roots, rosettes and flowers, although their expression levels appear to be lower than those of the five previously identified TOPP genes. Six of the eight TOPP genes were mapped to different positions on four of five A. thaliana chromosomes. Sequence comparison revealed that TOPP genes belong to different subgroups of plant PP1 genes, suggesting that they may encode proteins with distinct functions.     

Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses

We used the cytokinin-responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a beta-glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root-knot nematode (RKN) second-stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode-plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6-(Delta2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode-induced root galls per plant, than control hairy roots.     

The protein content of tissues in cynipid galls (Hymenoptera: Cynipidae): Similarities between cynipid galls and seeds

Cynipid galls are examples of induced plant development, where the gall inducer is in control of cell differentiation and morphogenesis of a new plant organ. This study concentrates on the tissues of the larval chamber common to all cynipid galls. The protein content of the inner gall tissue was compared to that of non‐gall plant tissues. We investigated three oak and two rose galls and their respective host plants. Total protein signatures of inner gall tissues were different from those of non‐gall plant tissues, and among the five galls. N‐terminal sequences were obtained for two abundant proteins from the inner gall tissues of D. spinosa and A. quercuscalicis, which were common to all galls, at 62 and 43 kDa. Database queries suggest the 62 kDa protein to be homologous to a protein disulphide isomerase (PDI), and the 43 kDa protein to be homologous to NAD‐dependent formate dehydrogenase (FDH). A naturally biotinylated protein was detected at 33 kDa during Western analyses with streptavidin. Western analyses revealed the presence of the biotinylated protein and PDI in the inner gall tissues of all five galls, while FDH was only detected in A. quercuscalicis and A. fecundator. PDI was also common to all non‐gall tissues, while FDH was not detected in non‐gall tissues, and the biotinylated protein was only detected in seeds. The proteins identified in the inner gall tissue suggest that (a) inner gall tissues in some galls are under respiratory stress, and (b) cynipid gall formation might involve the ectopic expression of seed‐specific proteins.