Molecular and bioinformatic analyses of nematode-derived proteins involved in plant-parasitism

Plant parasitic nematodes comprise several groups; the most economically damaging of these are the sedentary endoparasites. Sedentary endoparasitic nematodes modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Ultrastructural studies of plant-nematode interactions, and in particular those of the feeding sites, have identified two key feeding structures; feeding tubes and feeding plugs. Sedentary plant-parasitic nematodes feed by withdrawing host cell assimilate from the feeding site though a feeding tube. The function, composition and molecular characteristics of both feeding tubes and feeding plugs are poorly characterised. It is hypothesised that the apparent selective uptake of certain proteins from the feeding site is attributed to feeding tube size exclusion. A novel method is proposed to predict protein size based on protein database coordinates in silico. The validity of these predictions was tested using travelling wave ion mobility spectrometry – mass spectrometry, where predictions and measured values were within approximately 6% of each other. In silico predictions coupled with experimental techniques, such as mass spectrometry, analytical ultracentrifugation and protein electrophoresis, aimed to resolve seemingly-conflicting results of previous size exclusion experiments. Together these provided a pragmatic measurement of the upper limit for cyst nematode feeding tube size exclusion. Putative feeding-structure genes were identified from the genome sequence of the potato cyst nematode Globodera pallida using a series of reasoned assumptions about their characteristics. As a result, several large gene families were identified, one of which displayed highly complex genomic variation within a population. Subsequent characterisation of these candidate genes informed their function. The expression of several candidates was demonstrated in tissues with the capacity to secrete proteins into the host, implicating their role in host-pathogen interactions. In addition, for the 444 gene family, the protein was detected in the apoplasm, between the anterior end of the nematode and the feeding site. In planta host induced gene silencing targeting 444s reduced nematode infection by > 50%; further supporting their important role in successful parasitism. Feeding structure candidate genes were identified in de novo transcriptome assemblies of two related species (Globodera rostochiensis and Rotylenchulus reniformis). Differential expression analysis identified those candidates with congruent expression between species. 444 and 448 genes appear to be “core” effectors present in three genera of plant-parasitic nematodes that infect mono- and di-cotyledonous crop species

Sex: Not all that it's cracked up to be?

While sexual reproduction is generally thought to be, evolutionarily speaking, a good idea, there are a small number of organisms that are testament to the contrary. The root-knot nematodes Meloidogyne incognita, M. javanica, and M. arenaria reproduce clonally using mitotic parthenogenesis but have a broader host range, a wider geographical distribution, and a greater agricultural impact than their sexual relatives (Fig 1) [1]. Remarkably, some of these species even have the ability to overcome host resistance [2], suggesting a mechanism for adaptation in the absence of sex. The genetic basis of this plasticity, both in terms of host range and adaptability, is not fully understood. Previous genome sequencing of Meloidogyne has shown that the genome of one of these species, M. incognita, is polyploid [3], most likely as a result of hybridisation (allopolyploid), with a further study suggesting that M. incognita may be the result of multiple additive hybridisation events: a hybrid of a hybrid [4]

How Do Pathogens Evolve Novel Virulence Activities?

This article is part of the Top 10 Unanswered Questions in MPMI invited review series.We consider the state of knowledge on pathogen evolution of novel virulence activities, broadly defined as anything that increases pathogen fitness with the consequence of causing disease in either the qualitative or quantitative senses, including adaptation of pathogens to host immunity and physiology, host species, genotypes, or tissues, or the environment. The evolution of novel virulence activities as an adaptive trait is based on the selection exerted by hosts on variants that have been generated de novo or arrived from elsewhere. In addition, the biotic and abiotic environment a pathogen experiences beyond the host may influence pathogen virulence activities. We consider host-pathogen evolution, host range expansion, and external factors that can mediate pathogen evolution. We then discuss the mechanisms by which pathogens generate and recombine the genetic variation that leads to novel virulence activities, including DNA point mutation, transposable element activity, gene duplication and neofunctionalization, and genetic exchange. In summary, if there is an (epi)genetic mechanism that can create variation in the genome, it will be used by pathogens to evolve virulence factors. Our knowledge of virulence evolution has been biased by pathogen evolution in response to major gene resistance, leaving other virulence activities underexplored. Understanding the key driving forces that give rise to novel virulence activities and the integration of evolutionary concepts and methods with mechanistic research on plant-microbe interactions can help inform crop protection.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Characterisation of arabinogalactan endo β 1,4 galactanases from Globodera rostochiensis, Globodera pallida and Rotylenchulus reniformis

KL was funded by a BBSRC EASTBIO DTP studentship provided through the UKRI Biotechnology and Biological Sciences Research Council (BBSRC) grant number BB/T00875X/1 and by the Rural and Environment Science and Analytical Services Division of the Scottish Government at The James Hutton Institute and The University of St Andrews. Work on plant-parasitic nematodes at the University of Cambridge is supported by DEFRA licence 125034/359149/3 and funded by BBSRC grants BB/R011311/1, BB/N021908/1, and BB/S006397/1.Plant parasitic nematodes need to overcome the barrier presented by the plant cell wall in order to invade their host. A variety of plant cell wall degrading enzymes are present in endoparasitic nematodes including enzymes that degrade cellulose (beta 1,4 endoglucanases) and various pectin components. We describe the cloning and functional analysis of genes encoding GH53 arabinogalactan endo-1,4-beta-galactosidases from three related plant parasitic nematodes Globodera rostochiensis, Globodera pallida and Rotylenchulus reniformis. Phylogenetic and structural analyses strongly indicate that these genes have been acquired by horizontal gene transfer from bacteria. We show that the genes are expressed at invasive stages of the parasites in the secretory gland cells. We also demonstrate that the enzymes from these species are biochemically active, showing the expected hydrolytic enzymatic activity when galactan was used as a substrate. This work further demonstrates the importance of cell wall degradation to the success of the parasitic process and the extensive role that horizontal gene transfer has played in the evolution of plant parasitism by nematodes.Publisher PDFPeer reviewe

Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism

The draft genome of Ditylenchus dipsaci.

Ditylenchus dipsaci is a devastating pest to many crops worldwide. We present the first genome sequence for this species, produced with PacBio sequencing and assembled with CANU. Ditylenchus dipsaci is a devastating pest to many crops worldwide. We present the first genome sequence for this species, produced with PacBio sequencing and assembled with CANU

Functional C-terminally Encoded Peptide (CEP) plant hormone domains evolved de novo in the plant parasite Rotylenchulus reniformis

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and The James Hutton Institute through a PhD studentship to SE-vdA. The James Hutton Institute receives funding from the Scottish Government Rural and Environment Science and Analytical Services division. SE-vdA is supported by BBSRC grant BB/M014207/1.Sedentary Plant-Parasitic Nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re-differentiate into unique and metabolically active “feeding sites”. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C-terminally Encoded Peptide (CEP) plant hormone mimics (RrCEPs), in the syncytia-forming plant-parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP-encoding nematode genus identified to date (Meloidogyne), this suggests CEPs likely evolved de novo in R. reniformis. We have characterised the first member of this large gene family (RrCEP1), demonstrating its significant upregulation during the plant-nematode interaction and expression in the effector-producing pharyngeal gland cell. All internal CEP domains of multi-domain RrCEPs are followed by di-basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of upregulating plant nitrate transporter (AtNRT2.1) expression, while simultaneously reducing primary root elongation. When a non-CEP containing, syncytia-forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP-rich environment a smaller feeding site is produced. We hypothesise that CEPs of R. reniformis represent a two-fold adaptation to sustained biotrophy in this species; 1) increasing host nitrate uptake while 2) limiting the size of the syncytial feeding site produced.Publisher PDFPeer reviewe

STATAWAARS: a promoter motif associated with spatial expression in the major effector-producing tissues of the plant-parasitic nematode Bursaphelenchus xylophilus.

BACKGROUND: Plant-parasitic nematodes cause severe damage to a wide range of crop and forest species worldwide. The migratory endoparasitic nematode, Bursaphelenchus xylophilus, (pinewood nematode) is a quarantine pathogen that infects pine trees and has a hugely detrimental economic impact on the forestry industry. Under certain environmental conditions large areas of infected trees can be destroyed, leading to damage on an ecological scale. The interactions of B. xylophilus with plants are mediated by secreted effector proteins produced in the pharyngeal gland cells. Identification of effectors is important to understand mechanisms of parasitism and to develop new control measures for the pathogens. RESULTS: Using an approach pioneered in cyst nematodes, we have analysed the promoter regions of a small panel of previously validated pharyngeal gland cell effectors from B. xylophilus to identify an associated putative regulatory promoter motif: STATAWAARS. The presence of STATAWAARS in the promoter region of an uncharacterized gene is a predictor that the corresponding gene encodes a putatively secreted protein, consistent with effector function. Furthermore, we are able to experimentally validate that a subset of STATAWAARS-containing genes are specifically expressed in the pharyngeal glands. Finally, we independently validate the association of STATAWAARS with tissue-specific expression by directly sequencing the mRNA of pharyngeal gland cells. We combine a series of criteria, including STATAWAARS predictions and abundance in the gland cell transcriptome, to generate a comprehensive effector repertoire for B. xylophilus. The genes highlighted by this approach include many previously described effectors and a series of novel "pioneer" effectors. CONCLUSIONS: We provide a major scientific advance in the area of effector regulation. We identify a novel promoter motif (STATAWAARS) associated with expression in the pharyngeal gland cells. Our data, coupled with those from previous studies, suggest that lineage-specific promoter motifs are a theme of effector regulation in the phylum Nematoda.SE-vdA is supported by BBSRC grant BB/M014207/1 and BB/R011311/

Identification of candidate effector genes of <i>Pratylenchus penetrans</i>

Pratylenchus penetrans is one of the most important species of root lesion nematodes (RLNs) because of its detrimental and economic impact in a wide range of crops. Similar to other plant‐parasitic nematodes (PPNs), P. penetrans harbours a significant number of secreted proteins that play key roles during parasitism. Here, we combined spatially and temporally resolved next‐generation sequencing datasets of P. penetrans to select a list of candidate genes aimed at the identification of a panel of effector genes for this species. We determined the spatial expression of transcripts of 22 candidate effectors within the oesophageal glands of P. penetrans by in situ hybridization. These comprised homologues of known effectors of other PPNs with diverse putative functions, as well as novel pioneer effectors specific to RLNs. It is noteworthy that five of the pioneer effectors encode extremely proline‐rich proteins. We then combined in situ localization of effectors with available genomic data to identify a non‐coding motif enriched in promoter regions of a subset of P. penetrans effectors, and thus a putative hallmark of spatial expression. Expression profiling analyses of a subset of candidate effectors confirmed their expression during plant infection. Our current results provide the most comprehensive panel of effectors found for RLNs. Considering the damage caused by P. penetrans, this information provides valuable data to elucidate the mode of parasitism of this nematode and offers useful suggestions regarding the potential use of P. penetrans‐specific target effector genes to control this important pathogen