Molecular analysis of migratory plant-parasitic nematodes with a focus on plant cell wall modifying enzymes

Plant-parasitic nematodes cause a considerable amount of crop damage worldwide. These nematodes all have a stylet to damage plant cells mechanically, but through which they also secrete proteins important for their infection process. Most studies on genes encoding these so-called parasitism proteins were conducted on sedentary plant-parasitic nematodes which manipulate the plant to induce a specialized nematode feeding site. In this doctoral thesis parasitism genes of the less specialized migratory endoparasitic nematodes were investigated. The focus was on plant cell wall modifying enzymes that are being secreted by the nematode to enable migration through the rigid plant cell wall. The main component of the plant cell wall is cellulose, the most abundant sugar polymer on earth. In nematodes, cellulases or endo-1,4-β-glucanases which can degrade cellulose were mainly identified in sedentary species. A large part of this thesis is about the characterization of endoglucanases in migratory endoparasitic nematodes, namely in the species Radopholus similis, Ditylenchus africanus and Pratylenchus coffeae. Additionally, a detailed model for the evolution of the gene structure of endoglucanases within nematodes was proposed. The second most abundant sugar polymer in a plant cell wall is xylan. An endoxylanase from R. similis putatively able to degrade xylan was characterized. When this gene was downregulated in nematodes by RNAi, the infection on the plant was reduced by 60%, showing that the xylanase is important for the nematodes to infect their host plant. Another technique to identify parasitism genes is expressed sequence tag analysis, by randomly sequencing pieces of genes that are being expressed by the nematode. This approach was applied to D. africanus and 4847 ESTs were analyzed. Scientific research sometimes leads to unexpected discoveries. During an EST analysis of R. similis several ESTs were found with significant similarity to sequences from a Wolbachia endosymbiotic bacterium. This finding was further investigated and the presence of an intracellular Wolbachia bacterium in the nematode was demonstrated. Although this bacterium is often found in filarial nematodes, it is the first time it is discovered in a plant-parasitic nematode. Future research will be needed to determine what role this endosymbiont plays within the nematode

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event

Background Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included. Results Two new endoglucanases from the migratory nematodes Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea); all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida). Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes. Conclusions We conclude that the ancestral PPN GHF5 endoglucanase gene most probably consisted of the whole gene cassette, i.e. the GHF5 catalytic domain and the CBM2, rather than that it evolved by domain shuffling. Our evolutionary model for the gene structure in PPN GHF5 endoglucanases implies the occurrence of an early duplication event, and more recent gene duplications at genus or species level

Silencing of Mg-pat-10 and Mg-unc-87 in the plant parasitic nematode Meloidogyne graminicola using siRNAs

Until recently, the standard method for RNA interference (RNAi)-based reverse genetics in plant parasitic nematodes (PPNs) was based on the use of long double-stranded RNA (dsRNA). This increased the chance of off-target gene silencing through interactions between different short interfering RNAs (siRNAs) and non-cognate mRNA targets. In this work, we applied gene-specific knockdown of Mg-pat-10 and Mg-unc-87 of the root knot nematode Meloidogyne graminicola, using discrete 21 bp siRNAs. The homologue of Mg-pat-10 in C. elegans encodes body wall troponin C, which is essential for muscle contraction, whereas the homologue of Mg-unc-87 encodes two proteins involved in maintenance of the structure of myofilaments in the body wall muscle of C. elegans. The knockdown at the transcript level, as seen by semi-quantitative RT-PCR analysis, indicates that the Mg-pat-10 gene was silenced after soaking the nematodes in a specific siRNA for 48 h. At 72 h post-soaking, the Mg-pat-10 mRNA level was similar to the control, indicating the recovery of expression between 48 h and 72 h post-soaking. For Mg-unc-87 the nematodes started to recover from siRNA silencing 24 h after thorough washing. A migration assay showed that for the nematodes that were soaked in the control (siRNA of β-1,4-endoglucanase), 77% of the nematodes completed migration through the column in a 12 h period. By comparison with the control, nematodes incubated in the siRNA of pat-10 or unc-87 were significantly inhibited in their motility. After 12 h, only 6.3% of the juveniles incubated in the Mg-pat-10 siRNA and 9.3% of those incubated in Mg-unc-87 siRNA had migrated through the column, representing 91.8% and 87.9% inhibition respectively compared to the control. In the present work, we demonstrated that M. graminicola is readily susceptible to siRNAs of two genes involved in nematode motility. This is an important contribution to the progressive use of siRNA for functional analysis. Moreover, the application of RNAi in PPNs opens the way for environmentally friendly control of M. graminicola

Whole-genome deep sequencing reveals host-driven in-planta evolution of Columnea Latent Viroid (CLVd) quasi-species populations

Columnea latent viroid (CLVd) is one of the most serious tomato diseases. In general, viroids have high mutation rates. This generates a population of variants (so-called quasi-species) that co-exist in their host and exhibit a huge level of genetic diversity. To study the population of CLVd in individual host plants, we used amplicon sequencing using specific CLVd primers linked with a sample-specific index sequence to amplify libraries. An infectious clone of a CLVd isolate Chaipayon-1 was inoculated on different solanaceous host plants. Six replicates of the amplicon sequencing results showed very high reproducibility. On average, we obtained 133,449 CLVd reads per PCR-replicate and 79 to 561 viroid sequence variants, depending on the plant species. We identified 19 major variants (>1.0% mean relative abundance) in which a total of 16 single-nucleotide polymorphisms (SNPs) and two single nucleotide insertions were observed. All major variants contained a combination of 4 to 6 SNPs. Secondary structure prediction clustered all major variants into a tomato/bolo maka group with four loops (I, II, IV and V), and a chili pepper group with four loops (I, III, IV and V) at the terminal right domain, compared to the CLVd Chaipayon-1 which consists of five loops (I, II, III, IV and V)

Redirection of auxin flow in Arabidopsis thaliana roots after infection by root-knot nematodes

Plant auxin efflux and influx proteins redirect the plant hormone auxin towards the feeding site upon root-knot nematode infection in Arabidopsis thaliana roots.Plant-parasitic root-knot nematodes induce the formation of giant cells within the plant root, and it has been recognized that auxin accumulates in these feeding sites. Here, we studied the role of the auxin transport system governed by AUX1/LAX3 influx proteins and different PIN efflux proteins during feeding site development in Arabidopsis thaliana roots. Data generated via promoter-reporter line and protein localization analyses evoke a model in which auxin is being imported at the basipetal side of the feeding site by the concerted action of the influx proteins AUX1 and LAX3, and the efflux protein PIN3. Mutants in auxin influx proteins AUX1 and LAX3 bear significantly fewer and smaller galls, revealing that auxin import into the feeding sites is needed for their development and expansion. The feeding site development in auxin export (PIN) mutants was only slightly hampered. Expression of some PINs appears to be suppressed in galls, probably to prevent auxin drainage. Nevertheless, a functional PIN4 gene seems to be a prerequisite for proper nematode development and gall expansion, most likely by removing excessive auxin to stabilize the hormone level in the feeding site. Our data also indicate a role of local auxin peaks in nematode attraction towards the root

Tapping into the maize root microbiome to identify bacteria that promote growth under chilling conditions

Background When maize (Zea mays L.) is grown in the Northern hemisphere, its development is heavily arrested by chilling temperatures, especially at the juvenile phase. As some endophytes are beneficial for plants under stress conditions, we analyzed the impact of chilling temperatures on the root microbiome and examined whether microbiome-based analysis might help to identify bacterial strains that could promote growth under these temperatures. Results We investigated how the maize root microbiome composition changed by means of 16S rRNA gene amplicon sequencing when maize was grown at chilling temperatures in comparison to ambient temperatures by repeatedly cultivating maize in field soil. We identified 12 abundant and enriched bacterial families that colonize maize roots, consisting of bacteria recruited from the soil, whereas seed-derived endophytes were lowly represented. Chilling temperatures modified the root microbiome composition only slightly, but significantly. An enrichment of several chilling-responsive families was detected, of which the Comamonadaceae and the Pseudomonadaceae were the most abundant in the root endosphere of maize grown under chilling conditions, whereas only three were strongly depleted, among which the Streptomycetaceae. Additionally, a collection of bacterial strains isolated from maize roots was established and a selection was screened for growth-promoting effects on juvenile maize grown under chilling temperatures. Two promising strains that promoted maize growth under chilling conditions were identified that belonged to the root endophytic bacterial families, from which the relative abundance remained unchanged by variations in the growth temperature. Conclusions Our analyses indicate that chilling temperatures affect the bacterial community composition within the maize root endosphere. We further identified two bacterial strains that boost maize growth under chilling conditions. Their identity revealed that analyzing the chilling-responsive families did not help for their identification. As both strains belong to root endosphere enriched families, visualizing and comparing the bacterial diversity in these communities might still help to identify new PGPR strains. Additionally, a strain does not necessarely need to belong to a high abundant family in the root endosphere to provoke a growth-promoting effect in chilling conditions

Comparative genome analysis of pathogenic and non-pathogenic Clavibacter strains reveals adaptations to their lifestyle

BACKGROUND: The genus Clavibacter harbors economically important plant pathogens infecting agricultural crops such as potato and tomato. Although the vast majority of Clavibacter strains are pathogenic, there is an increasing number of non-pathogenic isolates reported. Non-pathogenic Clavibacter strains isolated from tomato seeds are particularly problematic because they affect the current detection and identification tests for Clavibacter michiganensis subsp. michiganensis (Cmm), which is regulated with a zero tolerance in tomato seed. Their misidentification as pathogenic Cmm hampers a clear judgment on the seed quality and health. RESULTS: To get more insight in the genetic features linked to the lifestyle of these bacteria, a whole-genome sequence of the tomato seed-borne non-pathogenic Clavibacter LMG 26808 was determined. To gain a better understanding of the molecular determinants of pathogenicity, the genome sequence of LMG 26808 was compared with that of the pathogenic Cmm strain (NCPPB 382). The comparative analysis revealed that LMG 26808 does not contain plasmids pCM1 and pCM2 and also lacks the majority of important virulence factors described so far for pathogenic Cmm. This explains its apparent non-pathogenic nature in tomato plants. Moreover, the genome analysis of LMG 26808 detected sequences from a plasmid originating from a member of Enterobacteriaceae/Klebsiella relative. Genes received that way and coding for antibiotic resistance may provide a competitive advantage for survival of LMG 26808 in its ecological niche. Genetically, LMG 26808 was the most similar to the pathogenic Cmm NCPPB 382 but contained more mobile genetic elements. The genome of this non-pathogenic Clavibacter strain contained also a high number of transporters and regulatory genes. CONCLUSIONS: The genome sequence of the non-pathogenic Clavibacter strain LMG 26808 and the comparative analyses with other pathogenic Clavibacter strains provided a better understanding of the genetic bases of virulence and adaptation mechanisms present in the genus Clavibacter. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-392) contains supplementary material, which is available to authorized users

Contamination Sources and Transmission Routes for Campylobacter on (Mixed) Broiler Farms in Belgium, and Comparison of the Gut Microbiota of Flocks Colonized and Uncolonized with Campylobacter

Biosecurity seems to be the most promising tool for Campylobacter control on poultry farms. A longitudinal molecular epidemiological study was performed during two production cycles, in which the broilers, the poultry house, and the environment of 10 (mixed) broiler farms were monitored weekly. Cecal droppings from the second production cycle were also used for 16S metabarcoding to study the differences in the microbiota of colonized and uncolonized flocks. Results showed that 3 out of 10 farms were positive for Campylobacter in the first production cycle, and 4 out of 10 were positive in the second. Broilers became colonized at the earliest when they were four weeks old. The majority of the flocks (57%) became colonized after partial depopulation. Before colonization of the flocks, Campylobacter was rarely detected in the environment, but it was frequently isolated from cattle and swine. Although these animals appeared to be consistent carriers of Campylobacter, molecular typing revealed that they were not the source of flock colonization. In accordance with previous reports, this study suggests that partial depopulation appears to be an important risk factor for Campylobacter introduction into the broiler house. Metabarcoding indicated that two Campylobacter-free flocks carried high relative abundances of Megamonas in their ceca, suggesting potential competition with Campylobacter