植物寄生线虫效应蛋白功能分析方法的研究进展

植物寄生线虫在侵染寄主过程中分泌许多与寄生相关的蛋白,这一类蛋白称为效应蛋白,这些效应蛋白在植物细胞内发挥各种作用,从而有利于线虫侵染、寄生和生长发育。研究这些效应蛋白的功能对于掌握线虫侵染植物的分子机理非常重要,也是寻找新的植物线虫病害防治方法的理论基础。对目前应用于研究植物寄生线虫效应蛋白功能的主要方法进行了概述。     

The plant apoplasm is an important recipient compartment for nematode secreted proteins

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode.     

RNAi in Haemonchus contortus: a potential method for target validation

RNA interference (RNAi) is a method for the functional analysis of specific genes, and is particularly well developed in the free-living nematode Caenorhabditis elegans. There have been several attempts to apply this method to parasitic nematodes. In a recent study undertaken in Haemonchus contortus, Geldhof and colleagues concluded that, although a mechanism for RNAi existed, the methods developed for RNAi in C. elegans had variable efficacy in this parasitic nematode. The potential benefits of RNAi are clear; however, further studies are required to characterize the mechanism present in parasitic nematodes, and to improve culture systems for these nematodes to monitor the long-term effects of RNAi. Only then could RNAi become a reliable assay of gene function.     

RNA interference and plant parasitic nematodes

RNA interference (RNAi) has recently been demonstrated in plant parasitic nematodes. It is a potentially powerful investigative tool for the genome-wide identification of gene function that should help improve our understanding of plant parasitic nematodes. RNAi should help identify gene and, hence, protein targets for nematode control strategies. Prospects for novel resistance depend on the plant generating an effective form of double-stranded RNA in the absence of an endogenous target gene without detriment to itself. These RNA molecules must then become available to the nematode and be capable of ingestion via its feeding tube. If these requirements can be met, crop resistance could be achieved by a plant delivering a dsRNA that targets a nematode gene and induces a lethal or highly damaging RNAi effect on the parasite.     

Recent progress in the development of RNA interference for plant parasitic nematodes

RNA interference (RNAi), first described for Caenorhabditis elegans , has emerged as a powerful gene silencing tool for investigating gene function in a range of organisms. Recent studies have described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when preparasitic juvenile nematodes take up double-stranded (ds)RNA that elicits a systemic RNAi response. Important developments over the last year have shown that in planta expression of a dsRNA targeting a nematode gene can successfully induce silencing in parasitizing nematodes. When the targeted gene has an essential function, a resistance effect is observed paving the way for the potential use of RNAi technology to control plant parasitic nematodes.     

Four transthyretin-like genes of the migratory plant-parasitic nematode Radopholus similis: members of an extensive nematode-specific family

Screening 1154 ESTs from the plant-parasitic nematode Radopholus similis resulted in seven tags coding for proteins holding a transthyretin-like domain (PF01060). The seven ESTs corresponded to four different genes which were cloned from a cDNA library (accession numbers AM691117, AM691118, AM691119, AM691120). Transthyretin-like genes belong to a large family, different from the transthyretin and the transthyretin-related genes with whom they share some sequence similarity at the protein level. This similarity has caused an inconsistent use of different names and abbreviations in the past. To avoid further confusion, we introduce a standardized nomenclature for this gene family, and chose to name this barely characterized gene family ttl (as for transthyretin-like). Further examination of the identified genes, named Rs-ttl-1 to -4, showed that they are expressed in both juveniles and adults, but not in young embryos. Whole mount in situ hybridization revealed a distinct spatial expression pattern for two of the genes: Rs-ttl-1 is expressed in the tissues surrounding the vulva, whereas Rs-ttl-2 is expressed in the ventral nerve cord. The deduced protein sequences contain a putative signal peptide for secretion, pointing to an extracellular function of the mature proteins. Database screens showed that the ttl family is restricted to nematodes. Moreover, a HMMER search revealed that ESTs derived from ttl genes are more abundant in parasitic nematode libraries, with a bias towards the parasitic stages. Despite their abundance in nematodes, including the extensively studied model organism Caenorhabditis elegans, the function of TTL proteins remains obscure. Our data suggest a role in the nervous system. Even without insight into their biological function, the nematode-specific nature of this gene family makes it a promising target for nematicides or RNAi mediated control strategies against parasitic nematodes.     

Research collaborations can improve the use of organic amendments for plant-parasitic nematode management

The concept of utilizing organic amendments to manage plant-parasitic nematodes is not new, but the widespread implementation of this management practice has still not been realized. The use of organic amendments for plant-parasitic nematode management is a complex process requiring an understanding of the transformation and generation of active compounds. As a result, research endeavors to understand and maximize the use of this management practice require a multidisciplinary approach which draws upon the expertise of nematologists, microbiologists, natural product chemists and soil scientists. Factors that require analysis and clarification include lethal concentration levels of organic amendments necessary to kill nematodes; chemical composition of incorporation material; fate and exposure potential to nematodes of compounds released into the soil; and the influence of environmental factors (i.e., temperature, microbial community, soil type) on the activity of organic amendments. Examples of research conducted in a collaborative manner with rye (Secale cereale) and a biosolid amendment demonstrate the power of multidisciplinary research. Only through collaborative research can consistent and reliable nematode suppression with organic amendments be achieved.     

Overview of organic amendments for management of plant-parasitic nematodes, with case studies from Florida

Organic amendments have been widely used for management of plant-parasitic nematodes. Relatively rapid declines in nematode population levels may occur when decomposing materials release toxic compounds, while longer-term effects might include increases in nematode antagonists. Improved crop nutrition and plant growth following amendment use may lead to tolerance of plant-parasitic nematodes. Results depend on a great variety of factors such as material used, processing/composting of material, application rate, test arena, crop rotation and agronomic practices, soil type, climate, and other environmental factors. Reasons for variable performance and interpretation of results from amendment studies are discussed. Case studies of amendments for nematode management are reviewed from Florida, where composts and crop residues are the most frequently used amendments. Plant growth was often improved by amendment application, free-living nematodes (especially bacterivores) were often stimulated, but suppression of plant-parasitic nematodes was inconsistent. Amendments were generally not as effective as soil fumigation with methyl bromide for managing root-knot nematodes (Meloidogyne spp.), and often population levels or galling of root-knot nematodes in amended plots did not differ from those in non-amended control plots. While amendments may improve plant growth and stimulate soil food webs, additional study and testing are needed before they could be used reliably for management of plant-parasitic nematodes under Florida conditions.     

Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection

Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets.     

宁南山区不同植被恢复方式下土壤线虫群落特征:形态学鉴定与高通量测序法比较

线虫是土壤食物网的重要组分, 也是土壤健康与生态系统恢复的重要指示生物, 因此准确测定线虫群落特征是发挥其生态指示作用的基础。传统线虫学研究多采用形态学鉴定方法, 但高通量测序技术近年来也逐渐受到重视。然而, 关于这两种方法的对比研究目前仍比较缺乏。本研究同时采用形态学鉴定和高通量测序法, 在黄土高原宁夏南部山区, 对不同植被恢复方式下(农田、自然恢复草地、柠条(Caragana korshinskii)人工林地和苜蓿人工草地)土壤线虫的数量、群落格局和生态指数进行了测定和比较。结果表明: (1)高通量测序仅能提供线虫类群的相对多度, 而形态学鉴定法能够测定土壤线虫的绝对多度, 后者测定结果表明3种植被恢复样地, 特别是自然恢复草地和柠条人工林地, 较农田具有更高的土壤线虫多度; (2)高通量测序法检获的线虫类群数(3纲4目26科42属)高于形态学鉴定法的测定结果(2纲3目18科27属), 但两种方法仅检获15个共有线虫属, 前者检测到的植物寄生线虫属数(22属)较后者(7属)显著增加, 而食细菌线虫和杂食-捕食线虫则相反; (3)在两种方法下, 相比农田, 3种植被恢复样地尤其是自然恢复草地和柠条人工林地, 其食微线虫的相对多度均显著下降, 而植物寄生线虫和杂食-捕食线虫的相对多度大幅上升, 这也导致线虫成熟度指数(MI)和植物寄生线虫指数(PPI)的提高及瓦斯乐卡指数(WI)的显著下降; (4)相比形态学鉴定法, 高通量测序法能检测到更丰富多样的植物寄生线虫, 在该方法下土壤线虫群落的组成、结构和生态指数在植被恢复样地与农田之间的差异也更为显著。综上所述, 采用形态学鉴定和高通量测序法测定的不同植被恢复方式下的线虫群落特征具有显著差异。     

Detection and Description of Soils with Specific Nematode Suppressiveness

Soils with specific suppressiveness to plant-parasitic nematodes are of interest to define the mechanisms that regulate population density. Suppressive soils prevent nematodes from establishing and from causing disease, and they diminish disease severity after initial nematode damage in continuous culturing of a host. A range of non-specific and specific soil treatments, followed by infestation with a target nematode, have been employed to identify nematode-suppressive soils. Biocidal treatments, soil transfer tests, and baiting approaches together with observations of the plant-parasitic nematode in the root zone of susceptible host plants have improved the understanding of nematode-suppressive soils. Techniques to demonstrate specific soil suppressiveness against plant-parasitic nematodes are compared in this review. The overlap of studies on soil suppressiveness with recent advances in soil health and quality is briefly discussed. The emphasis is on methods (or criteria) used to detect and identify soils that maintain specific soil suppressiveness to plant-parasitic nematodes. While biocidal treatments can detect general and specific soil suppressiveness, soil transfer studies, by definition, apply only to specific soil suppressiveness. Finally, potential strategies to exploit suppressive soils are presented.     

Alternatives to Fenamiphos for Management of Plant-Parasitic Nematodes on Bermudagrass

Plant-parasitic nematodes can be very damaging to turfgrasses. The projected cancellation of the registration for fenamiphos in the near future has generated a great deal of interest in identifying acceptable alternative nematode management tactics for use on turfgrasses. Two field experiments were conducted to evaluate the effectiveness of repeated applications of several commercially available nematicides and root biostimulants for reducing population densities of plant-parasitic nematodes and (or) promoting health of bermudagrass in nematode-infested soil. One experimental site was infested with Hoplolaimus galeatus and Trichodorus obtusus, the second with Belonolaimus longicaudatus. In both trials, none of the experimental treatments reduced population densities (P ≤ 0.1) of plant-parasitic nematodes, or consistently promoted turf visual performance or turf root production. Nematologists with responsibility to advise turf managers regarding nematode management should thoroughly investigate the validity of product claims before advising clientele in their use.     

The Surface Coat of Plant-Parasitic Nematodes: Chemical Composition,Origin, and Biological Role—A Review

Chemical composition, origin, and biological role of the surface coat (SC) of plant-parasitic nematodes are described and compared with those of animal-parasitic and free-living nematodes. The SC of the plant-parasitic nematodes is 5-30 nm thick and is characterized by a net negative charge. It consists, at least in part, of glycoproteins and proteins with various molecular weights, depending upon the nematode species. The lability of its components and the binding of human red blood cells to the surface of many tylenchid plant-parasitic nematodes, as well as the binding of several neoglycoproteins to the root-knot nematode Meloidogyne, suggest the presence of carbohydrate-recognition-domains for host plants and parasitic or predatory soil microorganisms (Pasteuria penetrans and Dactylaria spp., for example). These features may also assist in nematode adaptations to soil environments and to plant hosts with defense mechanisms that depend on reactions to nematode surfaces. Surface coat proteins can be species and race specific, a characteristic with promising diagnostic potential.     

Parasitism proteins in nematode-plant interactions

The current battery of candidate parasitism proteins secreted by nematodes to modify plant tissues for parasitism includes cell-wall-modifying enzymes of potential prokaryotic origin, multiple regulators of host cell cycle and metabolism, proteins that can localize to the plant cell nucleus, potential suppressors of host defense, mimics of plant molecules, and a relatively large cadre of predicted novel nematode parasitism proteins. Phenotypic effects of expressing nematode parasitism proteins in transformed plant tissues, protein-protein interaction assays, and RNA-mediated interference (RNAi) analyses are currently providing exciting evidence of the biological role of candidate nematode secreted parasitism proteins and identifying potential novel means of developing transgenic resistance to nematodes in crops.